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Contents
Timetable ··································································································································· 2
Floor Plan ··································································································································· 3
Scientific Programs ···················································································································· 4
Plenary Lectures ······················································································································ 15
PL1 ······································································································································ 17
PL2 ······································································································································ 20
Symposia ································································································································· 21
S1 ········································································································································· 23
S2 ········································································································································· 34
S3 ········································································································································· 40
S4 ········································································································································· 49
S5 ········································································································································· 56
S6 ········································································································································· 60
S7 ········································································································································· 68
S8 ········································································································································· 76
S9 ········································································································································· 85
S10 ······································································································································· 92
S11 ······································································································································· 99
S12 ····································································································································· 106
S13 ····································································································································· 114
Colloquium ······························································································································· 123
Workshop ······························································································································· 129
Poster Sessions ····················································································································· 133
Author Index ··························································································································· 209
The Microbiological Society of Korea
Rm. 810 (New Bldg.), The Korea Science & Technology Center 635-4, Yeoksam-dong,
Gangnam-gu, Seoul 135-703, Korea
Tel: +82-2-3453-3321/86
Fax: +82-2-3453-3322
Email: [email protected]
Homepage: www.msk.or.kr
This work was supported by the National Research Foundation of Korea Grant funded by the Korean
Government[NRF-2009-028-C00044].
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Timetable
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Floor Plan
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Session Room
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Taebaek Hall
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Session Room
Apricot Room
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Plenary Lectures
PL1
Plenary Lecture 1
May 6 (Thu.), Art Hall (B2)
Chair: Kye-Joon Lee, Seoul National University (Emeritus Professor)
17:30-18:15
Excellent DNA Polymerase from Thermococcus kodakaraensis KOD1 and
Its Application for PCR
Tadayuki Imanaka, Ritsumeikan University, Japan
PL2
Plenary Lecture 2
May 7 (Fri.), Art Hall (B2)
Chair: Doo-Hyun Nam, Yeungnam University
14:00-14:45
Post-transcriptional Regulation of Virulence Gene Expression in
Pseudomonas aeruginosa
Stephen Lory, Harvard Medical School, USA
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Symposia
S1
Bacterial Communications to the Environments
May 6 (Thu), Art Hall (B2)
Chair: Hyung-Yeel Kahng, Sunchon National University
S1-1
09:00-09:30
Exploration Untapped Halophilic Filamentous Actinomycetes:
Opportunity and Challenge
Wen-Jun Li, Yunnan University, China
S1-2
09:30-10:00
Viral Metagenomics and Phage Genomics
Jin-Woo Bae, Kyung Hee University
S1-3
10:00-10:30
Diversity and Biogeographic View of Thermophilic Archaea Isolated from
Terrestrial Hot Springs
Takashi Itoh, RIKEN BioResource Center, Japan
S1-4
10:30-11:00
Environmental Modulations and Progressive Selections in Dynamics of Genotypic
and Ecotypic Microdiversities of Coastal Vibrio Populations
Young-Gun Zo, Kyungsung University
S1-5
11:00-11:30
Geobacter for Harvesting Electricity from Waste Organic Matter and Uranium
Bioremediation
Byoung-Chan Kim, Korea Research Institute of Bioscience and Biotechnology
S2
Gene Expression and Growth Control
May 6 (Thu), Apricot Room (B1)
Chair: Sa-Ouk Kang, Seoul National University
S2-1
09:00-09:30
Potassium Mediates Escherichia coli Enzyme IIANtr-Dependent Regulation of
Sigma Factor Selectivity
Yeong-Jae Seok, Seoul National University
S2-2
09:30-10:00
Biochemical Characterization and Mutational Analysis of Fur-Family Proteins
from Bacillus licheniformis
Jin-Won Lee, Hanyang University
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S2-3
10:00-10:30
Delicate Control of Mitotic Exit by Bfa1 Asymmetry in
Budding Yeast Saccaromyces cerevisiae
Kiwon Song, Yonsei University
S2-4
10:30-11:00
Chromatin Dynamics during Transcription
Eun-Jung Cho, Sungkyunkwan University
S2-5
11:00-11:30
Insertion of Transmembrane Helices into the Mitochondrial Inner Membrane :
the Rules of the Game
Joy (Hyun) Kim, Seoul National University
S3
Pathogenesis of Clinically Important Bacterial Pathogens
May 6 (Thu), Baegam Hall (1F)
Chair: Myung-Je Cho, Gyeongsang National University
S3-1
09:00-09:30
Stimulatory Effect of Cell Elongation on Biofilm Formation in
Pseudomonas aeruginosa under Anaerobic Growth Condition
Sang Sun Yoon, Yonsei University
S3-2
09:30-10:00
Vibrio vulnificus Biofilm: Regulation and Roles of Extracellular Polysaccharides
Kyu-Ho Lee, Hankuk University of Foreign Studies
S3-3
10:00-10:30
Helicobacter pylori Infection in the Korean Population: An Epidemiological Link
Between Toxin Polymorphism and Disease
Douglas Scott Merrell, Uniformed Services University of the Health Sciences, USA
S3-4
10:30-11:00
Entry Mechanism and Intracellular Localization of Orientia tsutsugamushi into
Nonphagocytic Cell
Myung Sik Choi, Seoul National University
S3-5
11:00-11:30
Distinct Pathogenesis of Mycobacterium abscessus Isolated from Patients with
Upper Lobe Fibrocavitary Form of Pulmonary Disease
Sung Jae Shin, Chungnam National University
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S4
Dokdo's Biodiversity in the East Sea of Korea
(GIMB Session 1)
May 6 (Thu.), Art Hall (B2)
Chair: Jung-Sook Lee, Korea Research Institute of Bioscience and Biotechnology
S4-1
13:30-13:45
A Sustainable Research and Development on Dokdo
Hyun Soo Rho, East Sea Research Institute, KORDI
S4-2
13:45-14:00
The Study of Species Composition and Molecular Plant Geography in
Dokdo Island
Jae-Hong Pak, Kyungpook National University
S4-3
14:00-14:15
Marine Invertebrate Fauna of Dokdo and Molecular Phylogeography of
Sea Slaters and Chitons in Korea and Japan
Ui Wook Hwang, Kyungpook National University
S4-4
14:15-14:30
Exploring Microbial Functional Diversity of the Abyssal Seafloor in the East Sea
Jong-Shik Kim, Gyeongbuk Institute for Marine Bio-Industry (GIMB)
14:30-15:00
Discussion and Q & A
S5
Joint Symposium on Microorganism Resources Center
May 6 (Thu.), Baegam Hall (1F)
Organized by the Korea National Environmental Microorganisms Bank
Chair: Sang Seob Lee, Kyonggi University
S5-1
13:30-14:00
Practical Application of Environmental Microorganisms as Bioresources
Sang Seob Lee, Kyonggi University
S5-2
14:00-14:30
Current Status and Future Plan of KCTC
Jung-Sook Lee, Korea Research Institute of Bioscience and Biotechnology
S5-3
14:30-15:00
Management of Microbial Genetic Resources in Korean Agricultural Culture
Collection
In-Cheol Park, Rural Development Administration
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S6
Viruses and Immune Responses
May 6 (Thu.), Art Hall (B2)
Chair: Sin-Hyeog Im, Gwangju Institute of Science and Technology
S6-1
15:00-15:30
Molecular Signature of DC Subsets during Acute vs. Chronic Virus Infection
Sang-Jun Ha, Yonsei University
S6-2
15:30-16:00
Options for Control of Pandemic Influenza: Active and Passive Immunization
Huan Huu Nguyen, International Vaccine Institute
S6-3
16:00-16:30
Chromatin Remodeling, HIV Reservoir, and Drug Development for Eradication
of Chronic HIV Infection
Kee-Jong Hong, Korea National Institute of Health
S6-4
16:30-17:00
Automated HTS/HCS for Antivirals Using Visual HIV Full Replication Assays
Peter Sommer, Institut Pasteur Korea
S6-5
17:00-17:30
Probiotics as an Immune Modulator - Mechanism of Action and Therapeutic Applications
Sin-Hyeog Im, Gwangju Institute of Science and Technology
S7
Molecular Food Microbiology
May 6 (Thu.), Apricot Room (B1)
Chair: Jae-heon Kim, Dankook University
S7-1
15:00-15:35
Adaptive Cellular Survival Response to Oxidative and Inflammatory Stresses
Induced by Helicobacter pylori
Young-Joon Surh, Seoul National University
S7-2
15:35-16:10
Physiological Functionalities of Small Peptides
Toshiro Matsui, Kyushu University, Japan
S7-3
16:10-16:45
Anti-inflammatory Compounds from Medicinal Plants
Jae-Ha Ryu, Sookmyung Women's University
S7-4
16:45-17:20
Extraction of Bioactive Materials from Rice Hulls
Seung-Cheol Lee, Kyungnam University
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S8
Response to Environmental Cues
May 6 (Thu.), Baegam Hall (1F)
Chair: Hyoung Tae Choi, Kangwon National University
S8-1
15:00-15:30
Differential Roles of Yeast Glutamate Dehydrogenase Isozymes in Maintaining
Resistance to Stress-Induced Apoptosis
Pil Jae Maeng, Chungnam National University
S8-2
15:30-16:00
Cadmium Regulates Copper Homeostasis by Inhibiting Mac1 Activity,
a Transcriptional Activator of Copper Regulon, in Saccharomyces cerevisiae
Cheol-Won Yun, Korea University
Chair: Hyejoo Lee, Dong-A University
S8-3
16:00-16:30
Membrane Potential Changes by Proton Pumping Microbial Rhodopsins
Kwang-Hwan Jung, Sogang University
S8-4
16:30-17:00
Different Role of the DosS and DosT Histidine Kinases in Mycobacterial Response
to Hypoxic Conditions
Jeong-Il Oh, Pusan National University
S9
Design and Fabrication of Minimal Synthetic Cells
May 7 (Fri.), Art Hall (B2)
Chair: Ki-Sung Lee, Pai Chai University
S9-1
10:00-10:30
Synthetic 5′-Untraslated Regions for Fine-Tunable and Predictable Gene
Expression in Escherichia coli
Gyoo Yeol Jung, Pohang University of Science and Technology
S9-2
10:30-11:00
Rewriting Biology with Gene Synthesis
Duhee Bang, Yonsei University
S9-3
11:00-11:30
From Oligos to Organisms
Mikkel Algire, J. Craig Venter Institute, USA
S9-4
11:30-12:00
Artificial Assembly of a Minimal Cell
Giovanni Murtas, Enrico Fermi Centre, Italy
S9-5
12:00-12:30
Toward the Bacterial Minimal Chromosome
Young-Chang Kim, Chungbuk National University
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S10
Omics Study on the Marine Hyperthermophilic Archaea
(GIMB Session 2)
May 7 (Fri.), Baegam Hall (1F)
Sponsored by Marine & Extreme Genome Research Center, KORDI
Chair: Sung-Jae Lee, Kyung Hee University
S10-1 10:00-10:30
Novel Enzymes and Metabolic Pathways in Hyperthermophilic Archaea
Haruyuki Atomi, Kyoto University, Japan
S10-2 10:30-11:00
Global Proteome Analysis of Sulfur-Reducing Hyperthermophilic Archaeon
Thermococcus onnurineus NA1
Jong-Soon Choi, Korea Basic Science Institute
S10-3 11:00-11:30
Transcriptome Analysis on a Heat Shock Regulon in the Hyperthermophilic
Archaeon, Thermococcus kodakaraensis
Tamotsu Kanai, Kyoto University, Japan
S10-4 11:30-12:00
Molecular Architecture and the Mechanics of Lon, a Protease-Chaperone
Machine
Sun-Shin Cha, Korea Ocean Research & Development Institute (KORDI)
S10-5 12:00-12:30
Novel DNA Ligases with Broad Nucleotide Cofactor Specificity from the
Hyperthermophilic Crenarchaeota
Suk-Tae Kwon, Sungkyunkwan University
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S11
Pseudomonas Toxins and Pathogenicity
May 7 (Fri.), Art Hall (B2)
Chair: Joon-Hee Lee, Pusan National University
S11-1 14:45-15:15
In Situ Fluorescent Imaging of Bacteriogenic Cyanide in Lungs of Live Mice
Infected with Pseudomonas aerugionosa and Burkholderia cepacia
Sungsu Park, Ewha Womans University
S11-2 15:15-15:45
Iron Homeostasis Affects Antibiotic-Mediated Bacterial Cell Death
Woojun Park, Korea University
Chair: Woojun Park, Korea University
S11-3 15:45-16:15
Pseudomonas Virulence by a Quorum-Dependently Secreted Exoprotease
Joon-Hee Lee, Pusan National University
S11-4 16:15-16:45
Apoptotic Elimination of Natural Killer Cells by Pseudomonas aeruginosa
Jin Woong Chung, Dong-A University
S12
Protein Structure and Function
May 7 (Fri.), Apricot Room (B1)
Chair: Chankyu Park, Korea Advanced Institute of Science and Technology
S12-1 14:45-15:15
Structural Basis for Hypoxia Sensing by Histidine Kinases from
Mycobacterium tuberculosis
Beom Sik Kang, Kyungpook National University
S12-2 15:15-15:45
Structural Studies of the Transmembrane Domain of Bacterial Histidine Kinase
Young Ho Jeon, Korea Basic Science Institute
S12-3 15:45-16:15
Functional Characterization of Native and Recombinant Antifreeze Protein of
Arctic yeast, KOPRI-AY30
Hak Jun Kim, Korea Polar Research Institute
Chair: Kwang-Hwan Jung, Sogang University
S12-4 16:15-16:45
From Oxylipin to Volatile
Dongsun Lee, Jeju National University
S12-5 16:45-17:15
Spectroscopic Study of Rhodopsins in Action
Hideki Kandori, Nagoya Institute of Technology, Japan
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S13
Marine Biotechnology
(GIMB Session 3)
May 7 (Fri.), Baegam Hall (1F)
Chair: Kae Kyoung Kwon, Korea Ocean Research & Development Institute
S13-1
14:45-15:15
Marine Brown Kelp Diversity Based on the Analyses of Multigenes from Plastid
and Mitochondrion
Ga Youn Cho, National Institute of Biological Resources
S13-2
15:15-15:45
Functional Analysis in Escherichia coli of Biosynthesis Genes for Isoprenoids,
Carotenoids, and Sesquiterpenes, and Their Production with Higher Plants
Norihiko Misawa, Ishikawa Prefectural University, Japan
S13-3
15:45-16:15
5-Hydroxyindole-Type Alkaloids from Marine Organisms
Hyi-Seung Lee, Korea Ocean Research & Development Institute
S13-4
16:15-16:45
Marine Microbial Water Quality off Hawaii
Hans J. Krock, University of Hawai, USA (Emeritus Professor)
S13-5
16:45-17:15
Plant Growth-Promoting Fungi from the Sand-Dune Plants in East Coast of
Korea
Jong-Guk Kim, Kyungpook National University
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Colloquium
C1
Young Scientists Session 1
May 7 (Fri.), Apricot Room (B1)
Chair: Jin-ho Lee, Kyungsung University
C1-1
10:00-10:25
Pyrene Metabolism and Enzymes Involved in
Novosphingobium pentaromativorans US6-1
Yuanrong Luo, Korea Ocean Research & Development Institute
C1-2
10:25-10:50
Microarray Analysis of Knockout Mutant LuxS relevant to
the Quorum Sensing Mechanism in Escherichia coli K-12
Su-Wan Son, Kyonggi University
C1-3
10:50-11:15
Osmotic Stress Inhibits the Cell Growth of Listeria monocytogenes by
the Downregulation of PTS Genes
Dongryeoul Bae, Mississippi State University, USA
C1-4
11:15-11:40
Direct Activation of Transcription of Fur-Positive Regulon by
Iron-Free Form of Fur Protein
Hyun-Jung Lee, Hankuk University of Foreign Studies
13
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Workshop
W1
Workshop : Chunlab, Inc.
May 7 (Fri.), Baegam Hall (1F)
Chair: Yu-Seok Oh, Chunlab, Inc.
12:30-14:00
Microbial Community Analysis Using Pyrosequencing
Jongsik Chun, CEO of Chunlab, Inc.
14
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2010 International Meeting of
the Microbiological Society of Korea
Plenary Lectures
15
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16
Plenary Lectures
PL-1
Excellent DNA Polymerase from Thermococcus kodakaraensis
KOD1 and Its Application for PCR
Tadayuki Imanaka
Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan
E-mail: [email protected]
We reflect on some studies on the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 and its
enzymes. This strain can grow at temperatures up to the boiling point and also represents one of the simplest
forms of life. In fact, we have determined the complete genome sequence, and found that around 2300 ORFs are
enough to support the life. As expected, all enzymes displayed remarkable thermostability, and we have
determined some of the basic principles that govern this feature. To our delight, many of the enzymes exhibited
unique biochemical properties and novel structures not found in mesophilic proteins. Here, I focus on DNA
polymerase that is useful in application.
Hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Novel and useful enzymes
J. Biol. Chem., 281(15): 10533-10539 (2006)
J. Bacteriol., 187(20): 7072-7080 (2005)
J. Bacteriol., 185, 1705-1711 (2003)
Appl. Environ. Microbiol., 68, 3925-3931 (2002)
J. Biol. Chem., 277, 31656-31662 (2002)
J. Bacteriol., 184, 3689-3698 (2002)
J. Bacteriol., 184, 3305-3312 (2002)
J. Bacteriol., 184, 777-784 (2002)
Genome analysis
Genome Res., 15(3): 352-363 (2005)
Transcriptome analysis
J. Bacteriol., 188(16):5915-5924 (2006)
J. Biol. Chem. Published online (2007)
Gene disruption system
J. Bacteriol., 185, 210-220 (2003)
Appl. Environ. Microbiol., 71(7):3889-3899 (2005)
J. Bacteriol. Published online (2007)
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The KOD DNA polymerase is one of the most efficient thermostable PCR enzymes exhibiting higher
accuracy and elongation velocity than any other commercially available DNA polymerases. The crystallographic
studies were also done. However, even when KOD DNA polymerase was used for PCR, troubles with
nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA
polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA
polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and beta-G1, to KOD
DNA polymerase were obtained. Exonulease activity measurement and epitope mapping revealed that the
epitope for 3G8 is located in conserved regions among (family B) DNA polymerases (Region II), and the
epitope for beta-G1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was
performed, the specificity of target gene amplification became much higher than in reactions without
monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kb).
<20 bp
<20 bp
Deep Vent
Pfu
Processivity (Bases/Reaction)
300 bp
KOD
0
100
Taq
200
61
Deep Vent
23
Pfu
25
300
400
Extension efficiency (Bases/sec)
138
KOD
0
50
100
150
200
Taq
Deep Vent
1.16
Pfu
7.39
0.63
Mutation frequency (%)
0.15
0.09
KOD
KOD-Plus
0
2
4
6
8
In this symposium, development of PCR enzymes including (a) Characterization of a new PCR enzyme,
KOD DNA polymerase, (b) Engineering of a new long and accurate (LA) PCR enzyme, (c) Improvement of
PCR by neutralizing monoclonal antibodies, and (d) Structural analysis of KOD DNA polymerase will be
presented.
18
Plenary Lectures
References
[1] Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR, M.
Takagi, M. Nishioka, H. Kakihara, M. Kitabayashi, H. Inoue, B. Kawakami, M. Oka & T. Imanaka, Appl.
Environ. Microbiol.,63, 4504-4510 (1997).
[2] Characterization and application for hot start PCR of neutralizing monoclonal antibodies against KOD
DNA polymerase, H. Mizuguchi, M. Nakatsuji, S. Fujiwara, M. Tagaki & T. Imanaka, J. Biochem., 126,
762-768 (1999).
[3] Crystallographic studies on family B DNA polymerase from hyperthermophilic archaeon Pyrococcus
kodakaraensis strain KOD1, H. Hashimoto, T. Matsumoto, M. Nishioka, T. Yuasa, S. Takeuchi, T. Inoue,
S. Fujiwara, M. Takagi, T. Imanaka & Y. Kai, J. Biochem., 125, 983-986 (1999).
[4] Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant
enzyme, M. Nishioka, H. Mizuguchi, S. Fujiwara, S. Komatsubara, M. Kitabayashi, H. Uemura, M. Takagi
& T. Imanaka, J. Biotechnol., 88, 141-149, (2001).
[5] Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1,
H. Hashimoto, M. Nishioka, S. Fujiwara, M. Takagi, T. Imanaka, T. Inoue, & Y. Kai. J. Mol. Biol., 306,
469-77 (2001).
[6] Evolution of PCR Enzymes: Towards a better PCR system based on a KOD DNA polymerase, T. Imanaka
& M. Takagi, J. Chin. Inst. Chem. Engrs., 32, 277-288, (2001).
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PL-2
Post-Transcriptional Regulation of Pseudomonas aeruginosa
Virulence Gene Expression
Stephen Lory
Department of Microbiology and Molecular Genetics, Harvard Medical School,
200 Longwood Avenue, Waren Alpert 363, Boston, MA 02115, USA
Bacteria that are common inhabitants of soil or water reservoirs cause some of the most serious infections of
humans. Moreover, the ability of these opportunistic pathogens to thrive in a wide range of environments often
depends on the activities of specialized signal transduction pathways and complex regulatory networks.
Pseudomonas aeruginosa can cause serious acute infections in immunocompromized individuals, or chronic
infections in patients with cystic fibrosis. We have recently uncovered a regulatory network that functions as a
molecular switch controlling the expression of hundreds of genes, including those encoding acute toxic proteins,
and in a reciprocal mode, the formation of biofilm determinants important for chronic infections. The switch
operates by controlling the reversible phosphorylation of GacS/GacA two component system, which regulates
transcription of only two genes, the small RNAs (sRNA) RsmZ and RsmY. The global impact of the
GacS/GacA system is therefore due to post-transcriptional activities of these sRNAs, acting by antagonizing the
binding of the translational regulator RsmA to its target sites at the 5′ ends of transcripts. A number of gense
that are positively and negatively regulated by the RsmA, RsmY and RsmZ system were uncovered. This
post-translational regulatory mechanism allows bacteria to respond more rapidly to environmental changes than
by utilization of transcriptional regulatory systems.
20
Plenary Lectures
2010 International Meeting of
the Microbiological Society of Korea
Symposia
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www.msk.or.kr
22
Symposia
S1-1
Geobacter for Harvesting Electricity from Waste Organic Matter
and Uranium Bioremediation
Byoung-Chan Kim
Microbial Resource Center, Korean Collection for Type Cultures (KCTC),
Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Dissimilatory Fe(III)-reducing bacteria can obtain energy by coupling the oxidation of organic compounds to
the reduction of Fe(III). Molecular analysis of the composition of microbial communities has shown that the
Geobacteraceae, a family of dissimilatory Fe(III)-reducing bacteria in the delta subdivision of the class
Proteobacteria, are the predominant Fe(III)-reducing microorganisms in a wide variety of sedimentary
environments in which Fe(III) reduction is the principal terminal electron-accepting process. Geobacter species
can use alternate electron acceptors, such as the radionuclide U(VI) [1] and insoluble graphite electrodes [2].
Therefore, Geobacter species are important for harvesting electricity from waste organic matter and removing
uranium from groundwater. Geobacter species are also of interest because of their role in environmental
restoration. For example, Geobacter can destroy petroleum contaminants in polluted groundwater by oxidizing
these compounds to harmless carbon dioxide [3].
Recent studies have indicated that outer membrane c-type cytochromes and conductive nanowires (Geopilin)
are important for the electron transfer to Fe(III) and electrode by Geobacter sulfurreducens [4]. Elucidating
which cytochromes are involved in what type of reduction and electron transfer is not trivial because the
genome of G. sulfurreducens contains genes for over 110 c-type cytochromes, at least 30 of which are predicted
to be localized in the outer membrane where Fe(III) and electrode reduction is likely to take place. Notably, only
five of these outer membrane cytochromes have been found to be directly involved in electron transfer from
inside of cells to outside [5]. It also have been revealed that the outer surface cytochrome, OmcZ which is
localized in extra cellular biomatrix and the conductive nanowires are critical and essential for high-dense
electricity generation and important for the conductive biofilm formation by G. sulfurreducens [6]. Now there
are several evolved G. sulfurreducens strains including KN400 strain (TIME’s one of the 50 best inventions of
2009) that have been adapted on electrode [7]. These newly evolved electricigens are under investigation in
order to find what others are the key components for increasing efficiency of electron transfer by Geobacter
species.
References
[1] Lovley DR Nature reviews. Microbiology 1, 35 (2003).
[2] Lovley DR Nature reviews. Microbiology 4, 797 (2006).
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[3] Lovley DR Science 293, 1444 (2001).
[4] Reguera G, McCarthy KD, Mehta T, Nicoll JS, Tuominen MT, and Lovley DR Nature 435, 1098 (2005).
[5] Voordeckers JW, Kim BC, Izallalen M, and Lovley DR Applied and environmental microbiology, In press.
[6] Nevin KP, Kim BC, Glaven RH, Johnson JP, Woodard TL, Methé BA, Didonato RJ, Covalla SF, Franks
AE, Liu A, and Lovley DR PloS ONE 4(5), e5628 (2009).
[7] Yi H, Nevin KP, Kim BC, Franks AE, Klimes A, Tender LM, and Lovley DR Biosensors & bioelectronics
24, 3498 (2009).
24
Symposia
S1-2
Exploration Untapped Halophilic Filamentous Actinomycetes:
Opportunity and Challenge
Shu-Kun Tang1, Yun Wang2, Xiao-Yang Zhi1, Kai Lou2, Li-Li Zhang3,
Li-Xin Zhao1, Cheng-Lin Jiang1, Li-Hua Xu1, Chang-Jin Kim4, and Wen-Jun Li1*
1
The Key Laboratory for Microbial Resources of the Ministry of Education, P. R. China, and Laboratory for
Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology,
Yunnan University, Kunming, 650091, P. R. China
2
Xinjiang Institute of Microbiology, Xinjiang Academy of Agricultural Science, Urumqi,
Xinjiang, 830091, P. R. China
3
Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production &
Construction Corps, Tarim University, Alar, Xinjiang 843300, P. R. China
4
Functional Metabolite Research Center KRIBB, Daejeon 305-806
*Corresponding author: Wen-Jun Li, E-mail: [email protected]; [email protected]
Actinomycetes are still the most prolific producers of pharmacologically important compounds, accounting
for about 70% of the naturally derived antibiotics that are currently in clinical use (Bérdy, 2005). However, most
researches over the past decades were focusing on those actinomycetes in the general terrestrial soil environments,
such as soils, and sediments of the rivers or lakes, and thus induced re-discovering the redundancy strains
(special for members of the genera Streptomyces and Micromonospora) and well-known compounds.
Actinomycetal resources under extreme environments (including extreme high and low temperature, extreme
high or low pH, high salt concentration, etc) have received comparatively little attention from microbiologists.
Actinomycetes are regarded as one kind of sideline microorganisms and those under extreme environments are
better research materials for biological evolution and phylogenetic analysis. Moreover, these extremephilic
actinomycetes may have new functional genes and can produce new secondary metabolites, which can be used
for further new antibiotics screening. Since 1990s’, our research group has paid much attention to the research
on extremophilic actinomycetal resources. Here, we just report our research results on the diversity of halophilic
filamentous actinomycetes from three levels: species, functional genes and metabolites.
Since the first halophilic actinomycete Actinopolyspora halophila (Gochnauer et al., 1975) was reported,
people knew that some filamentous actinomycetes could grow and tolerate with high salt concentrations.
Meanwhile, in the past three decades, only few halophilic actinomycetes were discovered and cultured, which
included six species within three genera: Actinopolyspora halophila (Gochnauer et al., 1975), Actinopolyspora
motivallis (Yoshida et al., 1991), Actinopolyspora iraqiensis (Ruan et al., 1994), Nocardiopsis halophila
(Al-Tai and Ruan, 1994), Nocardiopsis kunsanensis (Chun et al., 2000) and Saccharomonospora halophila
Al-Zarban et al., 2002). The big problem is that scientists don’t know too much about their nutrient
requirements and ecological adaptive mechanisms, and they have no effective isolation methods, which are the
25
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main bottleneck to restrict the research development in this field.
Since the late 1990s’, our research group initiated the study on halophilic filamentous actinomycetes from
hypersaline environments, especially focusing on salt lakes in north-west China including Xinjiang and Qinghai
provinces. Based on our extensive studies on the biological characteristics of halophilic filamentous
actinomycetes (Tang et al., 2003), one efficient isolation medium (cellulose-casein multi-salts) (Tang et al.,
2008) was designed and applied specially for the isolation of filamentous halophilic actinomycetes in
hypersaline environments. By using this method, over 3000 halophilic filamentous actinomycete strains were
isolated from hypersaline soils or sediments of salt lakes in Xinjiang Province, China, and most of them are truly
halophilic, which have optimal NaCl growth concentrations, ranging from 5% to 15% (w/v), but not grow in
absent of NaCl.
Phylogenetic analysis based on 16S rRNA gene sequences revealed that isolated strains belong to fourteen
genera within seven families. In addition to the above mentioned three genera, there are one new suborder
Jiangellineae (Tang et al., 2010) with family Jiangellaceae (Tang et al., 2010), six new genera:
Streptomonospora (Cui et al., 2001), Myceligenerans (Cui et al., 2004), Haloactinospora (Tang et al., 2008),
Haloglycomyces (Guan et al., 2009), Haloechinothrix (Tang et al., 2010), Haloactinopolyspora (Tang et al.,
2010), and some newly reported halophilic actinomycete species in four known genera, which had never
halophilc strains discovered before: Prauserella halophila (Li et al., 2003, 2009); Amycolatopsis halophila
(Tang et al., 2010), Saccharopolyspora halophila (Tang et al., 2009), Georgenia halophila (Tang et al., 2010).
Our research results clearly indicated that there were so diverse halophilic filamentous actinomycetes, and the
high density of new or unknown actinomycetal resources in these untapped hypersaline extreme environments
totally overtook our expectation. Furthermore, our results also showed that the genera Nocardiopsis,
Saccharomonospora and Streptomonospora were the predominant groups of culturable halophilic filamentous
actinomycetes and they distributed widely in different saline environments, and until now none truly halophilic
Streptomyces and Micromonospora isolates were discovered in hypersaline environments in Xinjiang. Thus, on
the basis of our research results, we believe that microbial community in hypersaline environments is
completely different from that constitute of microbial community in general terrestrial environments, which
was generally predominated by genera Streptomyces and Micromonospora.
It is undoubtedly that new species will have new functional genes and new secondary metabolites, and will
certainly have new potential commercial use. Actinomycetes from hypersaline environments may be an
important source for discovery of new drugs. Thus, the specific PCR-based was preformed for further screening
from these isolated strains with PKS I, PKS II and NRPS functional genes. In addition, we also selected some
positive halophilic strains for further sequencing their functional gene sequences to explore their diversity and
difference with some other known from other environments. Moreover, large-scale batch fermentations were
performed for some selected positive strains for further deriving their secondary metabolites. Our research
results show that most halophilic filamentous actinomycete strains are positive for PKS I, PKS II, and NRPS
genes, their protein coding functional gene sequences are quite different from those actinomycetes from other
environments, and they also have quite different ecological and taxonomically characteristics, which clearly
26
Symposia
indicates that halophilic filamentous actinomycetes have huge potential capacity for producing novel antibiotics.
Our secondary metabolite research results can further confirm this conclusion. For example, Erythronolides H
and I, novel congeners of the clinically important antibacterial drug erythromycin A, have been isolated from
the new halophilic filamentous actinomycete strain, Actinopolyspora sp. YIM 90600. In addition to producing
the new erythromycin congeners, Actinopolyspora sp. YIM 90600 produces erythromycin C in a high titer. The
presence of the C-14 hydroxyl moiety and the C-6/C-18-epoxide in erythronolide H and the spiroketal moiety of
erythronolide I sheds new insights into structural diversity of erythromycin analog libraries potentially
accessible by combinatorial biosynthesis (Huang et al., 2009).
In conclusion, our route to discovering novel actinomycetal resources from those environments that have
been un- and under-explored extreme/neglected habitats is quite successful. Our research group not only
established the isolation method on halophilic filamentous actinomycetes, realized them from unculturalbe to
culturable, but also found their potential ability to produce new antibiotics. However, from the above inspiring
research results, we should be aware in our mind that it is not only an opportunity, but also a challenge brought
forward to us that how to make these extremophilic actinomycetal resources becoming practical antibiotic
products like scientists did before.
References
[1] Al-Tai, A. M. and Ruan, J. S. 1994. Nocardiopsis halophila sp. nov., a new halophilic actinomycete
isolated from soil. Int. J. Syst. Bacteriol. 44, 474-478.
[2] Al-Zarban, S. S., Al-Musallam, A. A., Abbas, I., Stackebrandt, E. and Kroppenstedt. R. M. 2002.
Saccharomonospora halophila sp. nov., a novel halophilic actinomycete isolated from marsh soil in
Kuwait. Int. J. Syst. Evol. Microbiol. 52, 555-558.
[3] Bérdy, J. 2005. Bioactive microbial metabolites. J. Antibiot. 58, 1-26.
[4] Chun, J., Bae, K. S., Moon, E. Y., Jung, S. O., Lee, H. K. and Kim, S. J. 2000. Nocardiopsis kunsanensis sp.
nov., a moderately halophilic actinomycete isolated from a saltern. Int. J. Syst. Evol. Microbiol. 50,
1909-1913.
[5] Cui, X-L., Mao, P-H., Zeng, M., Li, W-J., Zhang, L-P., Xu, L-H and Jiang, C-L. 2001. Streptimonospora
salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae. Int. J. Syst. Evol. Microbiol. 51,
357-363.
[6] Cui, X-L., Schumann, Stackebrandt, E., Kroppenstedt, R. M., Pukall, R., Xu, L-H., Rohde, M and Jiang,
C.L. 2004. Myceligenerans xiligouense gen. nov., sp. nov., a novel hyphae-forming member of the family
Promicromonosporaceae. Int. J. Syst. Evol. Microbiol. 54, 1287-1293.
[7] Gochnauer, M. B., Leppard, G. G., Komaratat, M. K., Novitsky, T. and Kushner, D. 1975. Isolation and
characterization of Actinopolyspora halophila, gen. et sp. nov., an extremely halophilic actinomycete. Can.
J. Microbiol. 21, 1500-1511.
[8] Guan, T-W., Tang, S-K., Wu, J-Y., Zhi X-Y., Li-Hua Xu, L. H., Zhang, L-L. and Li. W. J. 2009.
Haloglycomyces albus gen. nov., sp. nov., a novel halophilic filamentous actinomycete of the family
Glycomycetaceae. Int. J. Syst. Evol. Microbiol. 59, 1297-1301.
27
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[9] Huang, S-X., Zhao, L-X., Tang, S-K., Jiang, C-L., Duan, Y-W. and Shen, B. Erythronolides. 2009. H and I,
new erythromycin congeners from a new halophilic actinomycete Actinopolyspora sp. YIM90600.
Organic Letters 11, 1353-1356.
[10] Li. W. J., Xu, P., Tang, S-K., Xu, L-H., Kroppenstedt, R.M., Stackebrandt, E and Jiang, C-L. 2003.
Prauserella halophila sp.nov. and Prauserella alba sp. nov., two new moderately halophilic
actinomycetes isolated from the saline soil. Int. J. Syst. Evol. Microbiol. 53, 1545-1549.
[11] Li, Y., Tang S-K., Chen, Y-G., Wu, J-Y., Zhi X-Y., Zhang, Y-Q. and Li. W. J. 2009. Prauserella
salsuginis sp. nov., Prauserella flava sp. nov., Prauserella aidingensis sp. nov. and Prauserella sediminis
sp. nov., isolated from a salt lake. Int. J. Syst. Evol. Microbiol. 59, 2923-2928.
[12] Ruan, J. S., Al-Tai, A. M., Zhou, Z. H., and Qu, L. H. 1994. Actinopolyspora iraquiensis sp. nov., a new
halophilic actinomycete isolated from soil. Int. J. Syst. Bacteriol. 44, 759-763.
[13] Tang, S-K., Li. W. J., Zhang, R-G., Wang Dong, Li-Hua Xu, and Cheng-Lin Jiang. 2003. Studies on
biological characteristics of Some halophilic and halotolerant actinomycetes Isolated from saline and
alkaline soils. Actinomycetologica 17, 6-10.
[14] Tang, S-K., Tian, X-P., Zhi, X-Y., Cai, M., Wu, J-Y., Yang, L-L., Xu, LH. and Li. W-J. 2008.
Haloactinospora alba gen. nov., sp. nov., a halophilic filamentous actinomycete of the family
Nocardiopsaceae. Int. J. Syst. Evol. Microbiol. 58, 2075-2080.
[15] Tang, S-K., Wang, Y., Cai, M., Zhi, X-Y., Lou, K., Xu, L. H., Jiang, C. L. and Li, W. J. 2009.
Saccharopolyspora halophila sp. nov., a novel halophilic actinomycete isolated from a saline lake in China.
Int. J. Syst. Evol. Microbiol. 59, 555-558.
[16] Tang, S-K., Wang, Y., Guan, T-W., Lee, J-C., Kim, C-J. and Li, W-J. 2010. Amycolatopsis halophila sp.
nov., a novel halophilic actinomycete isolated from a salt lake in China. Int. J. Syst. Evol. Microbiol. (In
Press).
[17] Tang, S-K., Wang, Y., Lee, J-C., Lou, K., Park, D-J., Kim, C-J. and Li, W-J. 2010. Georgenia halophila sp.
nov., a novel halophilic actinobacterium isolated from a salt lake in China. Int. J. Syst. Evol. Microbiol. (In
Press).
[18] Tang, S-K., Wang, Y., Wu, J-Y., Cao, L-L., Lou, K., Xu, L-H., Jiang, C-L. and Li, W. J. 2009.
Saccharopolyspora qijiaojingensis sp. nov., a novel halophilic actinomycete isolated from a salt lake in
China. Int. J. Syst. Evol. Microbiol. 59, 2166-2170.
[19] Tang, S-K., Wang, Y., Zhang, H., Lee, J-C., Lou, K., Kim, C-J. and Li, W-J. 2010. Haloechinothrix alba
gen. nov., sp. nov., a novel halophilic filamentous actinomycete of the suborder Pseudonocardineae. Int. J.
Syst. Evol. Microbiol. (In Press).
[20] Tang, S-K., Zhi, X-Y., Wang, Y., Shi, R., Lou, K., Xu L-H. and Li, W-J. 2010 Haloactinopolyspora alba
gen. nov. sp. nov., a novel halophilic filamentous actinomycete isolated from a salt lake in China, with
proposal of Jiangellaceae fam. nov. and Jiangellineae subord. nov. Int. J. Syst. Evol. Microbiol. (In Press).
[21] Yoshida, M., Matsubara, K., Kudo, T. and Horikoshi, K. 1991. Actinopolyspora mortivallis sp. nov., a
moderately halophilic actinopmycete. Int. J. Syst. Bacteriol. 41, 15-20.
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Symposia
S1-3
Viral Metagenomics and Phage Genomics
Jin-Woo Bae*, Eun-Jin Park, and Kyoung-Ho Kim
Department of Life and Nanopharmaceutical Sciences and Department of Biology,
Kyung Hee University, Seoul 130-701
Although viruses are known to be the most numerous biological entities in soil and seawater, little is known
about their diversity in this environment. To investigate viruses in environment, we combined two kinds of
approaches, the culture-independent and the culture-dependent method. We used the viral metagenomics and
viral genomics to understand viral diversity and physiology. For the metagenomic approach, viruses were
separated and concentrated with centrifugation and filtration from a soil, foods, feces, and a marine environment.
Viral DNA was extracted and amplified with the multiple displacement amplification (MDA) method, a whole
genome amplification method which uses the phi29 DNA polymerase and random hexamer to amplify DNA
isothermally. The metagenomes amplified by MDA were sequenced and/or compared with sequences from
metagenomes amplified by the linker amplified shotgun library method (LASL) which amplified only double
strand DNA. The analysis of sequences showed that the MDA method amplify single stranded DNA viral
genomes more preferentially than other (mostly double stranded) viral DNA. Changes of circular DNA and
linear DNA amount during MDA were observed with quantitative real time PCR to confirm the preferential
amplification of circular DNA. As a result, we detected that various kinds of unknown single stranded DNA
viruses exit in soil and marine environment. We also could assemble several circular genomic compounds of
unknown putative single stranded DNA viruses from metagenomic sequences retrieved from soil and marine
environment.
We also obtained the metagenomic viromes in order to identify and characterize viral diversity and
community structure in fermented foods. Viral particles were purified and concentrated by sequential filtrations
and ultracentrifugation. Extracted DNA was amplified by the linker amplified shotgun library (LASL) method
and the amplified metagenomes were sequenced by 454 pyrosequencing. Moreover, in order to look more
deeply the biological entities and correlation of viral with bacterial communities in the fermented foods, we also
analyzed the bacterial community by pyrosequencing at the same time. Here, we report the first metagenomic
analyses to investigate the unknown viral communities from three fermented foods, fermented shrimp, kimchi,
and sauerkraut, fermented by the action of innate microorganisms. The story of metagenomic viromes gives us
to better understand biological ecosystems in the fermented foods.
References
[1] M. Breitbart, F. Rohwer, Trends Microbiol, 13, 278, 2005.
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[2] J. A. Fuhrman, U. Campbell, Nature, 393, 410,1998.
[3] F. Rohwer, R. Edwards, J Bacteriol, 184, 4529, 2002.
[4] T. Allander et al., Proc Natl Acad Sci USA, 102, 12891, 2005.
[5] A. F. Andersson et al., PLoS One, 3, e2836, 2008.
[6] M. Breitbart et al., J Bacteriol, 185, 6220, 2003.
[7] S. R. Gill et al., Science, 312, 1355, 2006.
[8] A. Lopez-Bueno et al., Science, 326, 858, 2009.
[9] S. G. Tringe et al., Science, 308, 554, 2005.
[10] P. J. Turnbaugh et al., Nature, 457, 480, 2009.
[11] P. J. Turnbaugh et al., Nature, 444, 1027, 2006.
[12] J. C. Venter et al., Science, 304, 66, 2004.
[13] T. Woyke et al., Nature, 443, 950, 2006.
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Symposia
S1-4
Diversity and Biogeographic View of Thermophilic Archaea Isolated
from Terrestrial Hot Springs
Takashi Itoh
Japan Collection of Microorganisms, RIKEN BioResource Center, Wako-shi, Saitama 351-0198, Japan
Thermophilic archaea would play important roles in promising areas of biosciences and biotechnologies: for
example, understanding of the adaptive machineries and contribution to biogeochemical cycles in
geothermally-heated environments, speculation of models to infer an early life form, and providing sources of
heat-stable enzymes. In order to support researches in these fields, RIKEN BRC-JCM has been collecting,
preserving, and distributing strains of the thermophilic archaea. Besides, we have isolated the thermophilic
archaea extensively from terrestrial hot springs in Japan and the Philippines to incorporate novel thermophilic
archaea into our collection. In this paper, I survey the phylogenetic positions of the archaeal isolates on the basis
of the 16S rRNA gene sequences to discuss their taxonomic diversity. In addition, a comparison between the
phylogenetic relationship and the geographic distribution of a certain group of the thermophilic archaea is made
to shed light on the speciation process.
Up to now, more than 190 archaeal strains were isolated from hot springs in Japan and the Philippines by
using modified media of the “Sulfolobus” medium. The 16S rRNA gene sequence analyses revealed that they
were distributed within five archaeal orders: Sulfolobales (131 isolates), Thermoproteales (36 isolates),
Acidilobales (12 isolates), Desulfurococcales (1 isolate), and Thermoplasmatales (11 isolates). In addition to
the genera published by ourselves (i.e., Thermocladium, Caldivirga, Vulcanisaeta, Caldisphaera, and
Thermogymnomonas), there were three hitherto undescribed genera in the order Sulfolobales, and at least eight
new species as predicted by the phylogenetic distance to known species (<98% similarity).
Among these isolates, members of the genus Vulcanisaeta seemed to be distributed endemically in view of
the 16S rRNA gene sequence comparisons. The genus Vulcanisaeta was created to encompass two rod-shaped,
hyperthermophilic arachaeal species, V. distributa and V. souniana (Itoh et al., 2002). The V. distributa strains,
which were all isolated from spouting hot spring or heated solfataric soil samples, are classified into the 16S
rRNA gene-based subgroups, which are congruent with the geographic locations as well. On the other hand, the
V. souniana strains, isolated from pipelined hot spring water, allocate in a different lineage. This may indicate
that the geographic separation and the environmental difference promote the genetic diversification, leading to
the speciation, among members of the genus Vulcanisaeta. In order to verify the hypothesis, we have conducted
the phylogenetic analyses on radA and DEAD/DEAH box helicase-like genes. These results would underpin the
usefulness of these Vulcanisaeta strains as model organisms to study the allopatric microbial speciation.
Reference
Itoh, T. et al., Int. J. Syst. Evol. Microbiol. 52: 1097-1104 (2002).
31
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S1-5
Environmental Modulations and Progressive Selections in Dynamics
of Genotypic and Ecotypic Microdiversities of Coastal Vibrio
Populations
Young-Gun Zo1* and Nipa Chokesajjawatee2
1
Department of Biology, Kyungsung University,
National Center for Genetic Engineering and Biotechnology, National Science and Technology Agency, Thailand
2
How bacterial populations respond to environmental fluctuations has been an intriguing question. There have
been three kinds of approaches employed to address this question, namely [1] relating fine-scale genotypic
variation of one or a few house-keeping genes to environmental variations using of a massive number of
bacteria present at a given geographical location, [2] relating genome-wide genotypic information of a limited
number of bacterial isolates to environmental variation, and [3] relating genotypic and phenotypic variations of
a census-collection of bacteria to spatio-temporal variations of environmental conditions from which the
collection was obtained. In this study, findings from these approaches were synthesized by regarding Vibrio
populations in coastal environments as a model case.
When massive collections of 16S rRNA gene sequences and gyrB were analyzed, the fine-scale diversity
comprising natural bacterial assemblages, i.e., microdiversity structure, appeared to be derived from a neutral
radiation of genetic traits and occasional selection. In this case, the dynamics of phylogenetic signals detected in
the DNA sequences from natural environments did not corresponded to environmental parameters [1-2].
However, the correspondence was found when genome-wide genetic information of Vibrio cholerae was
employed as the tool for genotyping [3-4]. Therefore, assessment of phenotypic correspondence from a large
number of bacterial isolates warranted the proper resolution from this conflict.
In this study, a set of V. cholerae isolates from a census of the species undertaken in the upper Chesapeake
Bay was employed in an analysis of its 25 phenotypic and genotypic traits. Two distinct V. cholerae types were
observed for every three isolates in the set. Results of time scale analysis indicated that turnover in the
operational taxonomic units of V. cholerae populations occurred monthly, whereas prevalence of bacterial traits
changed at three month intervals. Environmental variables, including pH, salinity and zooplankton composition
showed significant correlation with variation in both genotypic and phenotypic characteristics. Progressive
selective pressure on V. cholerae population structure in the natural environment is concluded to operate at the
fine scale of the ecological niche.
As evidence validating the progressive selection of fine-scale ecological niches, the unique energy-demanding
accessory trait of bioluminescence was examined. Succession from non-luminescent to luminescent populations
32
Symposia
of V. cholerae in coastal waters occurred during spring to mid-summer, along with a shift from neutral to
alkaline (pH>8) pH vales. The occurrence of an intermediate luminescent population that had phylogenetic
relatedness to non-luminescent populations peaked in the middle of the pH shift. It is concluded that each cluster
of luminescent V. cholerae occupy a distinct ecological niche.
In conclusion, active communication of bacterial cells with the environment appears to results in tight
coupling between slight changes in the environment and gradual succession in bacterial community. The
success is neither stochastic nor striking. These findings warrants that fine-scale variation in genotypic/
phenotypic characteristics of bacteria community is predictable based on some knowledge in environmental
variations.
References
[1] Acinas S.G., Klepac-Ceraj V., Hunt D.E., Pharino C., Ceraj I., Distel D.L., and Polz M.F. Nature, 430, 551,
2004.
[2] Thompson J.R., Pacocha S., Pharino C., Klepac-Ceraj V., Hunt D.E., Benoit J., Sarma-Rupavtarm R.,
Distel D.L., and Polz M.F. Science 307, 1311, 2005.
[3] Keymer D. P., Miller M.C., Schoolnik G.K., and Boehm A.B. Appl. Environ. Microbiol. 73, 3705, 2007.
[4] Miller M.C., Keymer D.P., Avelar A., Boehm A.B., and Schoolnik G.K. Appl. Environ. Microbiol. 73,
3695, 2007.
33
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S2-1
Potassium Mediates Escherichia coli Enzyme IIANtr-Dependent
Regulation of Sigma Factor Selectivity
Chang-Ro Lee1, Seung-Hyon Cho1, Hyun-Jin Kim1, Miri Kim1, Alan Peterkofsky2,
and Yeong-Jae Seok1,3*
1
Laboratory of Macromolecular Interactions, Department of Biological Sciences and Institute of Microbiology,
Seoul National University, Seoul 151-742
2
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,
MD 20892, USA
3
Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-742
While the intracellular concentration of Na+ does not exceed 20 mM, that of K+ is maintained at 300-500 mM
in Escherichia coli (1). A high cytoplasmic K+ concentration is required for processes such as maintenance of
cell turgor and adaptation of cells to osmotic conditions. Here we show that sigma factor selectivity is one of the
important reasons for the accumulation of such high concentrations of K+ in E. coli cytosol.
Recently, we have shown that an Escherichia coli K12 mutant devoid of enzyme IIANtr (EIIANtr) of the
nitrogen PTS becomes extremely sensitive to leucine or leucine-containing peptides due to the defect in
derepression of the ilvBN operon encoding acetohydroxy acid synthase I (2). The derepression mechanism was
turned out to be due to the interaction of unphosphorylated EIIANtr with TrkA, an essential component of the
Trk potassium transport system to decrease the intracellular potassium level (3). In this study, we report the
mechanism for regulation of gene expression by the intracellular K+ level. We report here the mechanism for
regulation of gene expression by the intracellular K+ level. The leucine hypersensitivity of a ptsN (encoding
EIIANtr) mutant was suppressed by deleting the rpoS gene, encoding the stationary phase σ factor. Despite
intracellular levels of sigma factors comparable to the wild-type strain, most of the genes down-regulated in a
ptsN mutant are controlled by σ70, while all the up-regulated genes are controlled by σS, implying that the
balance of sigma activities is modified by ptsN deletion. This change of sigma factor activity was found to be
due to increased levels of K+. In vitro transcription assays showed that a σ70 controlled gene and a σS controlled
gene were differentially affected by potassium concentration. Biochemical studies revealed that K+ is
responsible for sigma factor competition by differentially influencing the binding of σ70 and σS to core RNA
polymerase. Taken together, the data indicate that EIIANtr controls sigma factor selectivity by regulating the
intracellular K+ level.
References
[1] Bossemeyer, D., Borchard, A., Dosch, D. C., Helmer, G. C., Epstein, W., Booth, I. R., and Bakker, E. P.
(1989) J Biol Chem 264, 16403-16410.
34
Symposia
[2] Lee, C.-R., Koo, B.-M., Cho, S.-H., Kim, Y.-J., Yoon, M.-J., Peterkofsky, A., and Seok, Y.-J. (2005) Mol
Microbiol 58, 334-344.
[3] Lee, C.-R., Cho, S.-H., Yoon, M.-J., Peterkofsky, A., and Seok, Y.-J. (2007) Proc Natl Acad Sci USA 104,
4124-4129.
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S2-2
Biochemical Characterization and Mutational Analysis of
Fur-Family Proteins from Bacillus licheniformis
Chang-Jun Ji, Jung-Hoon Kim, and Jin-Won Lee*
Department of Life Science, Hanyang University, Seoul 133-791
The ferric uptake regulator (Fur) family proteins includes sensors of Fe (Fur), Zn (Zur), Mn (Mur), Ni (Nur),
and peroxide (PerR). Bacillus subtilis, a model Gram-positive bacterium, contains three Fur homologues: Fur,
Zur, and PerR. However, the genome sequence of Bacillus licheniformis indicates that there are two additional
Fur homologues, BL00690 and BL00950, in addition to Fur, Zur, and PerR homologues. We confirmed that the
fur, zur and perR homologous genes from B. licheniformis can complement the fur, zur and perR mutants
respectively, and that PerR from B. licheniformis can sense H2O2 by oxidation of two histidine residues like
PerR from B. subtilis. Although, untill now, no target genes have been identified for BL00690 and BL00950,
our biochemical and mutational analyses strongly indicate that both BL00690 and BL00950 contain one Zn
atom presumably coordinated by four cysteine residues and can sense H2O2 by histidine oxidation like PerR
protein.
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S2-3
Delicate Control of Mitotic Exit by Bfa1 Asymmetry in
Budding Yeast Saccaromyces cerevisiae
Kiwon Song
Dept. of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749
All eukaryotic cells become duplicated by a process called cell cycle. The key issue of cell cycle is to
maintain genomic integrity while duplicating itself. Chromosomes are replicated once and equally segregated as
daughter chromosomes at the anaphase of mitosis. Once chromosomes have segregated properly, a pathway
called mitotic exit network (MEN) becomes activated to finish mitosis. Timing of the MEN activation should be
tightly coordinated with proper segregation of the chromosomes to ensure genomic integrity. How the timing of
mitotic exit is controlled is a current topic of interest. In budding yeast Saccharomyces cerevisiae that provides
an excellent model to study mitotic exit, the Tem1 functions at the top of the MEN and Bfa1 negatively
regulates Tem1. The polo kinase Cdc5 also activates MEN by directly phosphorylating and inhibiting Bfa1. The
spindle pole body (SPB) acts as a platform for these MEN components. Bfa1/Bub2 complex localizes to SPBs
and Tem1 association with SPBs depends on Bfa1/Bub2. As the spindle aligns along the mother-bud axis to
segregate duplicated chromosomes in anaphase, Bfa1/Bub2 becomes exclusively present on the bud-oriented
SPB. Conversely, when spindles are misaligned, Bfa1/Bub2 is present on both SPBs and mitotic exit is delayed,
suggesting that the spatial distribution of Bfa1/Bub2 controls the timing of mitotic exit. However, the molecular
mechanism and function of how asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB)
during anaphase controls mitotic exit are not well understood, particularly in unperturbed cells. Here, we
identified novel Cdc5 target residues within the Bfa1 C-terminus, 452S,
453
S, 454S, and
559
S. A Bfa1 mutant in
4A
which all of these residues have been changed simultaneously (called Bfa1 ) persisted on both SPBs at
anaphase and was hypo-phosphorylated, despite retaining its GAP activity against Tem1. These observations
demonstrate a tight link between localization and phosphorylation, and no direct connection between
asymmetric localization and GAP activity of Bfa1. Consistent with this, in kinase-defective cdc5-2 cells, Bfa1
was unphosphorylated and localized to both SPBs. The BFA14A cells progressed through anaphase normally,
but displayed a delayed mitotic exit in unperturbed cell cycles. Altogether, we suggest that Cdc5 modulates the
asymmetric Bfa1 distribution to the bud-directed SPB independently of Bfa1 GAP activity at anaphase, and that
Bfa1 asymmetry fine-tunes the timing of MEN activation.
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S2-4
Chromatin Dynamics during Transcription
Eun-Jung Cho
Sungkyunkwan University
The eukaryotic genome is packed by formation of nucleosomes with histones H2A, H2B, H3, and H4. The
bulk of the nucleosomes are assembled when DNA is replicated in the S phase via the replication coupled (RC)
pathway, which is mediated by histone H3/H4 chaperones, CAF-1 and Asf1. Outside of the S phase, histones
are deposited into the nucleosome by HIR (mammalian homolog of HIRA) and Asf1 through a replication
independent (RI) pathway. Recently, considerable attention has been devoted to these deposition pathways
because RC or RI pathways are considered to be the potential mechanistic tool to sort out various types of
histones along the genome according to the biological function of chromatin.
My lab is interested in the role of the H3/H4 chaperones in the chromatin dynamics and wishes to determine
the chromatin homeostasis during transcription. We examined the role of histone chaperones and different
histone forms during transcription. The potential consequence of histone deposition via the RI pathway will be
discussed with the yeast model system.
38
Symposia
S2-5
Insertion of Transmembrane Helices into the Mitochondrial Inner
Membrane: the Rules of the Game
Joy (Hyun) Kim
Laboratory of Membrane Biology, School of Biological Sciences, Seoul National University, Seoul 151-747
While overall hydrophobicity is generally recognized as the main characteristic of transmembrane a-helices,
the only membrane system for which we have detailed quantitative data on how different amino acids contribute
to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells [1, 2, 3].
Here, we provide data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane
of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong
determinants of membrane insertion. These results parallel what has been found previously for the ER. However,
we see striking differences between the effects elicited by charged residues flanking the transmembrane
segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated
dissimilarity between the two insertion systems.
References
[1] Hessa, T. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature
433, 377-381, (2005).
[2] Hessa, T. et al. Molecular code for transmembrane-helix recognition by the Sec61 translocon.Nature 450,
1026-1030, (2007).
[3] Hessa, T., Reithinger, J. H., von Heijne, G. & Kim, H. Analysis of transmembrane helix integration in the
endoplasmic reticulum in S. cerevisiae. J Mol Biol 386, 1222-1228, (2009).
39
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S3-1
Stimulatory Effect of Cell Elongation on Biofilm Formation in
Pseudomonas aeruginosa under Anaerobic Growth Condition
Sang Sun Yoon
Department of Microbiology, Yonsei University College of Medicine, Seoul
Pseudomonas aeruginosa, a Gram-negative bacterium of clinical importance, forms robust biofilm under
anaerobic conditions that mimic the abnormally thickened mucus layer lining the airway of patients with cystic
fibrosis (CF). The molecular basis behind this enhanced biofilm formation is unknown. We identified a
morphological change naturally accompanied by anaerobic respiration in P. aeruginosa and investigated its
effect on biofilm formation. A standard laboratory strain, PAO1, was elongated during anaerobic respiration
compared to bacteria grown aerobically. The degree of cell elongation was dependent on the presence of nitrite
reductase (NIR) that reduces nitrite (NO2-) to nitric oxide (NO) and was repressed in PAO1 in the presence of
carboxy-PTIO, a NO antagonist. This demonstrated that cell elongation involves a process that responds to NO,
a spontaneous byproduct of anaerobic respiration. The non-elongated NIR-deficient mutant failed to form
biofilm, while the mutant of nitrate reductase (NAR) and PAO1, both of which were highly elongated, formed
robust biofilm. Furthermore, decreased biofilm formation was also observed in PAO1 anaerobically grown in
the presence of C-PTIO. Outer membrane protein, OprE, was highly upregulated during anaerobic respiration.
An ΔoprE mutant was not as elongated as PAO1 and formed defective biofilm. Moreover, OprE synthesis was
minimal in the non-elongated NIR-deficient mutant further supporting its vital role in cell elongation and
biofilm formation under anaerobic condition. Taken together, our data suggest that anaerobiosis-induced cell
elongation plays a critical role in biofilm formation.
40
Symposia
S3-2
Vibrio vulnificus Biofilm:
Regulation and Roles of Extracellular Polysaccharides
Han-Suk Kim and Kyu-Ho Lee*
Department of Environmental Science, Hankuk University of Foreign Studies
To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio
vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased
biofilm-forming activity was found to contain a transposon insertion in the ntrC gene encoding a well-known
transcriptional activator. We examined how this regulator modulates a biofilm-forming process in V. vulnificus
by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wildtype strains grown
under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD
(ADP-glycero-manno-heptose-6-epimerase) was elevated during the growth period for biofilm formation and
was strongly influenced by NtrC. A luxAB-transcriptional fusion with the gmhD promoter region indicated that
gmhD expression was positively regulated by NtrC. The function of the gmhD gene product in V. vulnificus was
assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in
production of mature lipopolysaccharide (LPS) and demonstrated an attenuated ability to form a biofilm. These
results suggest that NtrC acts as a key regulator of LPS biosyntheses and, thereby, modulates critical steps in
biofilm development of V. vulnificus.
In addition, the regulatory roles of NtrC in exopolysaccharide (EPS) biosynthesis were studied with three
gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and
showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes
(VV1_2661, VV2_1579, and VV1_2305) in each cluster showed decreased production of EPS, attenuated
ability to form biofilm, and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579
and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in
VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus,
and some EPS components play important roles in interacting with hosts.
References
[1] Grau, B.L., Henk, M.C., Garrison, K.L., Olivier, B.J., Schulz, R.M., O’Reilly, K.L., and Pettis, G.S. (2008)
Further characterization of Vibrio vulnificus rugose exopolysaccharide gene cluster. Infect Immun 76:
1485-1497.
[2] Joseph, L.A., and Wright, A.C. (2004) Expression of Vibrio vulnificus capsular polysaccharide inhibits
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biofilm formation. J Bacteriol 186: 889-893.
[3] Kim, H.-S., Lee, M.-A., Chun, S.-J., Park, S.-J., and Lee, K.-H. (2007) Role of NtrC in biofilm formation
via controlling expression of the gene encoding an ADP-glycero-manno-heptose-6-epimerase in the
pathogenic bacterium, Vibrio vulnificus. Mol Microbiol 63: 559-574.
[4] Kim, H.-S., S.-J. Park, and K.-H. Lee. (2009) Role of NtrC-regulated exopolysaccharides in the biofilm
formation and pathogenic interaction of Vibrio vulnificus. Molecular Microbiology. Mol Microbiol 74:
436-453.
[5] Lee, J.H., Rho, J.-B., Park, K.-J., Kim, C.-B., Han, Y.-S., Choi, S.H., Lee, K.-H., and Park, S.-J. (2004).
Role of flagellum and motility in pathogenesis of Vibrio vulnificus. Infect Immun 72: 4905-4910.
[6] Park, N.Y., Lee, J.H., Kim, M.W., Jeong, H.G., Lee, B.C., Kim, T.S., and Choi, S.H. (2006) Identification
of the Vibrio vulnificus wbpP gene and evaluation of its role in virulence. Infect Immun 74: 721-728.
[7] Yildiz, F.H., and Visick, K.L. (2009) Vibrio biofilms: so much the same yet so different. Trends Microbiol
17: 109-118.
42
Symposia
S3-3
Helicobacter pylori Infection in the Korean Population:
An Epidemiological Link Between Toxin Polymorphism and Disease
D. Scott Merrell, Ph.D.
Associate Professor, Uniformed Services University, Bethesda, MD 20814, USA
Helicobacter pylori is a medically important pathogen, and although infection rates vary geographically,
globally this bacterium colonizes over 50% of the world’s population (1, 2). This spiral shaped, Gram-negative,
microaerophilic bacterium chronically inhabits the unforgiving environment of the stomach, and causes
subclinical gastritis in the majority of patients. However, in some individuals, H. pylori colonization results in
peptic ulcer disease; 75% of gastric ulcers and 90% of duodenal ulcers are attributed to H. pylori infection (3).
In its most severe sequelae, H. pylori infection can lead to the development of two forms of gastric cancer:
adenocarcinoma and MALT lymphoma (4-7). The association of H. pylori with stomach cancer led the World
Health Organization to classify it as a class I carcinogen in 1994 (8). It currently remains the only bacterium to
obtain this perilous distinction.
H. pylori strains express various toxins that enable the bacteria to cause host cell damage. Included among these
toxins are the cytotoxin associated gene A (CagA) and the vacuolating cytotoxin (VacA) (9). CagA has emerged
as a major contributor to disease severity, and there is a direct link between presence of CagA and increased
cancer risk (10, 11). CagA induces various pathologic changes by modulating host cell signaling pathways,
primarily after tyrosine phosphorylation at the EPIYA motif (12-19). The most common motifs have been
designated as EPIYA-A, -B, -C, and -D (14), and are found in two distinct combinations by geographic location.
VacA is another important toxin that is produced and secreted by all H. pylori strains (20, 21), and has been
shown to have various modes of action (22-29). Like CagA, VacA has been shown to contain a number of
polymorphisms. Currently, three polymorphic regions of vacA have been identified: signal (s), intermediate (i),
and middle (m). Each of these polymorphic regions has two main types that divide them further into type 1 and
type 2 (30, 31). The s region encodes the N-terminal signal sequence (32, 33), and polymorphisms in the s
region affect anion channel-forming efficiency of the toxin (32). The s1 type has an increased ability to form
membrane channels (32). Polymorphisms in the m region affect the cell tropism of the toxin (34); the m1 type of
VacA shows toxicity to a broader range of cells than the m2 type (35, 36). The i region, located between the s
and m regions, also displays two main polymorphisms (30). The i1 type of VacA has stronger vacuolating
activity than the i2 type (30). Individually, the s1, i1, and m1 types have been shown to be associated with more
severe forms of H. pylori induced disease (30, 37).
Gastric cancer is the second most common cause of cancer death worldwide, and this fact could be reflective
43
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of the high incidence of H. pylori infection (38-41). Interestingly, geographic areas with the highest level of
gastric cancer, which include most East Asian countries, also have the highest rate of H. pylori infection (39, 40,
42). Additionally, in East Asian countries, 90% of strains carry cagA. Indeed, South Korea, has one of the
highest rates of H. pylori colonization (43) and one of the highest rates of gastric cancer in the world (11, 44).
Recently, we conducted a large-scale molecular epidemiologic analysis of South Korean strains, and found a
statistical link between the East Asian CagA, EPIYA-ABD genotype and development of gastric cancer.
Statistical analysis showed that the proportion of ABD genotype varied significantly by diagnosis (P=0.022),
and that this distribution was statistically different when compared to gastritis (P=0.004) or duodenal ulcer
patients (P=0.014). Characterization of a subset of the Korean isolates showed that all strains from cancer
patients expressed and delivered phosphorylateable CagA to host cells, whereas the presence of the cagA gene
did not strictly correlate to expression and delivery of CagA in all non-cancer strains. Moreover, we genotyped
the isolates for vacA, and then analyzed the data to determine if particular genotypes varied across disease state,
sex, or cagA allele. Of these strains, 206 strains carried a s1/i1/m1 allele, 11 strains carried a s1/i1/m2 allele, and
8 strains carried a s1/i2/m2 allele. A statistical association between variation in the cagA and vacA alleles was
identified (P=0.0007), and log linear modeling revealed that this variation affects severity of disease outcome
(P=0.027). Additionally, we found evidence that variation within the middle (m) region of VacA contributes
significantly to the distribution of vacA alleles across gender (P=0.008) as well as the association with disease
outcome (P=0.011). In the Korean population, the majority of H. pylori strains carry the vacA s1/i1/m1 allele
and the CagA EPIYA-ABD allele. These facts may contribute to the high incidence of gastric maladies
including gastric cancer within this population.
*Excerpts taken from Journal of Clinical Microbiology, 47(4):959-68, 2009 and Journal of Clinical
Microbiology, 48(2):559–567, 2010.
References
[1] The EUROGAST Study Group, Gut 34, 1672 (Dec, 1993).
[2] T. Matysiak-Budnik, F. Megraud, J Physiol Pharmacol 48 Suppl 4, 3 (Sep, 1997).
[3] P. B. Ernst, B. D. Gold, Annu Rev Microbiol 54, 615 (2000).
[4] M. J. Blaser, Bmj 316, 1507 (May 16, 1998).
[5] J. Parsonnet et al., N Engl J Med 330, 1267 (May 5, 1994).
[6] J. Parsonnet et al., N Engl J Med 325, 1127 (Oct 17, 1991).
[7] N. J. Talley et al., J Natl Cancer Inst 83, 1734 (Dec 4, 1991).
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Symposia
[8] International Agency for Research on Cancer., in Monographs on the Evaluation of Carcinogenic Risks to
Humans. (Lyon, 1994), vol. 61, pp. 177-240.
[9] C. Montecucco, R. Rappuoli, Nat Rev Mol Cell Biol 2, 457 (Jun, 2001).
[10] M. J. Blaser et al., Cancer Res 55, 2111 (May 15, 1995).
[11] J. Gwack et al., Br J Cancer 95, 639 (Sep 4, 2006).
[12] M. Hatakeyama, Oncogene 27, 7047 (Nov 24, 2008).
[13] H. Higashi et al., Science 295, 683 (Jan 25, 2002).
[14] H. Higashi et al., Proc Natl Acad Sci U S A 99, 14428 (Oct 29, 2002).
[15] H. Higashi et al., J Biol Chem 279, 17205 (Apr 23, 2004).
[16] B. G. Neel, H. Gu, L. Pao, Trends Biochem Sci 28, 284 (Jun, 2003).
[17] K. Roovers, R. K. Assoian, Bioessays 22, 818 (Sep, 2000).
[18] R. Tsutsumi, A. Takahashi, T. Azuma, H. Higashi, M. Hatakeyama, Mol Cell Biol 26, 261 (Jan, 2006).
[19] M. Hatakeyama, Nat Rev Cancer 4, 688 (Sep, 2004).
[20] T. L. Cover, S. R. Blanke, Nat Rev Microbiol 3, 320 (Apr, 2005).
[21] J. C. Atherton et al., J Clin Microbiol 37, 2979 (Sep, 1999).
[22] T. L. Cover, S. A. Halter, M. J. Blaser, Hum Pathol 23, 1004 (Sep, 1992).
[23] B. Gebert, W. Fischer, E. Weiss, R. Hoffmann, R. Haas, Science 301, 1099 (Aug 22, 2003).
[24] L. Manente et al., J Cell Physiol 214, 582 (Mar, 2008).
[25] R. Pai, T. L. Cover, A. S. Tarnawski, Biochem Biophys Res Commun 262, 245 (Aug 19, 1999).
[26] I. Szabo et al., EMBO J 18, 5517 (Oct 15, 1999).
[27] M. R. Terebiznik et al., Autophagy 5, 370 (Apr, 2009).
[28] V. J. Torres, S. E. VanCompernolle, M. S. Sundrud, D. Unutmaz, T. L. Cover, J Immunol 179, 5433 (Oct
15, 2007).
[29] D. C. Willhite, S. R. Blanke, Cell Microbiol 6, 143 (Feb, 2004).
[30] J. L. Rhead et al., Gastroenterology 133, 926 (Sep, 2007).
[31] J. C. Atherton et al., J Biol Chem 270, 17771 (Jul 28, 1995).
[32] M. S. McClain et al., J Bacteriol 183, 6499 (Nov, 2001).
[33] A. P. Pugsley, Microbiol Rev 57, 50 (Mar, 1993).
[34] X. Ji et al., Infect Immun 68, 3754 (Jun, 2000).
[35] C. Pagliaccia et al., Proc Natl Acad Sci U S A 95, 10212 (Aug 18, 1998).
[36] M. R. Amieva, E. M. El-Omar, Gastroenterology 134, 306 (Jan, 2008).
[37] D. Basso et al., Gastroenterology 135, 91 (Jul, 2008).
[38] A. I. Neugut, M. Hayek, G. Howe, Semin Oncol 23, 281 (Jun, 1996).
[39] K. D. Crew, A. I. Neugut, World J Gastroenterol 12, 354 (Jan 21, 2006).
[40] S. Yamamoto, Jpn J Clin Oncol 31, 471 (Sep, 2001).
[41] D. M. Parkin, F. Bray, J. Ferlay, P. Pisani, CA Cancer J Clin 55, 74 (Mar-Apr, 2005).
[42] Y. O. Ahn et al., J Korean Med Sci 6, 7 (Mar, 1991).
[43] S. Tokudome et al., Asian Pac J Cancer Prev 8, 462 (Jul-Sep, 2007).
[44] A. Shin et al., Br J Cancer 92, 1273 (Apr 11, 2005).
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S3-4
Entry Mechanism and Intracellular Localization of
Orientia tsutsugamushi into Nonphagocytic Cell
Myung Sik Choi*, Hyuk Chu, Sang Wook Kim, Nam Hyuk Cho, and Ik Sang Kim
Department of Microbiology and Immunology, Seoul National University College of Medicine
Orientia tsutsugamushi are obligate intracellular bacteria that grow within the cytoplams of the eukaryotic
host cell. Therefore its capability of the attachment and entry into the cell surface and intracellular localization
are critical steps in oriential pathogenesis.
O. tsutsugamushi enters the host cell via unknown mechanism, this intracellular bacterium needs to exploit
host endocytotic pathway. It has been reported that clathrin-mediated or caveolar-mediated endocytosis
pathways are the main targets for many other intracellular bacteria such as Chlamydia, Listeria, and Rickettisa.
To investigate the endocytotic pathway exploited by O. tsutsugamushi, we used several specific inhibitors to
block, either clathrin-mediated or caveolar-mediated endocytotic pathway. The inhibitors blocking
clathrin-mediated endocytotic pathway have clearly reduced the infectivity of O. tsutsugamushi into
nonprofessional phagocytic cells. In contrast, the inhibitors blocking caveolar-mediated endocytotic pathway
did not affect the infectivity. We further confirmed a localization of O. tsutsugamushi with clathrin or adaptor
protein 2 (AP-2) in clathrin coated vesicles.
In order to identify the endosomal escape of O. tsutsugamushi, colocalization of O. tsutsugamushi with
molecules that are expressed either in early endosome or late endosome in early endosome antigen 1 (EEA1)
and lysosomal-associated protein (LAMP2) was achieved. The result showed that orientia were colocalizaed
with the late endosomal-lysosomal glycoprotein, LAMP2, during an early infection, but within 2 h this
colocalization decreased significantly disappeared. This study suggested that O. tsutsugamushienter
nonprofessional phagocytic cells such as endotheilal cells and fibroblast through clathrin-mediated endocytotic
pathway and escapes from the endosome to the cytosol at phagosome/lysome state not ealry endosome.
Next, we examined the mechanism of intracellular localization using ECV 304 cells, endothelial cell line.
ECV 304 cells infected with O. tsutsugamushi recealed the collocation of microtubule organizing center
(MTOC) and cytosolic orientiae by indirect immunoflurescence assay. Using immunofluorescence microscopy
in presence and absence of MT depolymerizing agents (colchicine and nocodazole), it was shown that the
cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of p50/dynamitin,
which is known to associate with dynein-dependent movement), the movement of O. tsutsugamushi to the
MTOC was also mediated by dynein, the minus end directed MT-related motor. Although the significance of
this movement in the cycle of O. tsutsugamushi was not proven, we propose that the cytosolic O. tsutsugamushi
46
Symposia
uses MTs and dyneins to propel them from the cell periphery to the MTOC. This study is one of the few
examples of the use of MT network by a nonmembrane bounded particle or microorganism.
References
[1] SW Kim, KS Ihn, SH Han, SY Seong, IS Kim, and MS Choi. Microtubule- and Dynein Mediated
Movement of Orientia tsutsugamushi to the Microtubule Organizing Center Infection and immunity. 69,
494, 2001.
[2] H Chu, JH Lee, SH Han, SY Kim, NH Cho,IS Kim, and MS Choi. Exploitation of the Endocytic Pathway
by Orientia tsutsugamushi in Nonprofessional Phagocytes Infection and Immunity, 74, 4246, 2006.
47
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S3-5
Distinct Pathogenesis of Mycobacterium abscessus Isolated from
Patients with Upper Lobe Fibrocavitary Form of Pulmonary Disease
Sung Jae Shin*, Byung Soo Lee, Choul-Jae Won, Kwang-Wook Kim, Hyun Bae Kang,
and Hosung Sohn
Department of Microbiology and Infection Signaling Network Research Center, College of Medicine,
Chungnam National University, Daejeon 301-747
Mycobacterium abscessus is a member of the group of rapidly growing mycobacteria (RGM), which cause a
wide range of clinical diseases, including cutaneous disease, osteomyelitis, post-traumatic wound infection, and
chronic lung disease [1-4]. M. abscessus has been identified as the causative organism in approximately 80% of
cases of RGM pulmonary disease [1, 2]. In addition, M. abscessus is resistant to many antibiotics and thus is
very difficult to treat [2, 5]. Pulmonary disease caused by nontuberculous mycobacteria (NTM), including M.
abscessus, can be classified into two distinct types of clinical disease; the upper lobe fibrocavitary (UC) form
and nodular bronchiectatic (NB) form [6]. In a very recent publication, our group reported the differential
virulence between mycobacterial strains in accordance to the forms of the pulmonary M. abscessus diseases. In
present study, we further to discuss the distinct pathogenesis of clinical strains based on the recent analysis of
whole genome sequence , transposon mutants, and the pattern of macrophage cell death.
References
[1] Brown-Elliott BA and Wallace RJ Jr. Clinical and taxonomic status of pathogenic nonpigmented or
late-pigmenting rapidly growing mycobacteria. Clin Microbiol Rev 2002;15:716-46.
[2] Griffith DE, Girard WM, and Wallace RJ Jr. Clinical features of pulmonary disease caused by rapidly
growing mycobacteria. An analysis of 154 patients. Am Rev Respir Dis 1993;147:1271-8.
[3] Sanguinetti M, Ardito F, Fiscarelli E, La Sorda M, D'Argenio P, Ricciotti G, et al. Fatal pulmonary
infection due to multidrug-resistant Mycobacterium abscessus in a patient with cystic fibrosis. J Clin
Microbiol 2001;39:816-9.
[4] Wallace RJ Jr., Swenson JM, Silcox VA, Good RC, Tschen JA, Stone MS. Spectrum of disease due to
rapidly growing mycobacteria. Rev Infect Dis 1983;5:657-79.
[5] Jeon K, Kwon OJ, Lee NY, Kim BJ, Kook YH, Lee SH, et al. Antibiotic treatment of Mycobacterium
abscessus lung disease: a retrospective analysis of 65 patients. Am J Respir Crit Care Med 2009;
doi:10.1164/rccm.200905-0704OC.
[6] Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. ATS Mycobacterial
Diseases Subcommittee; American Thoracic Society; Infectious Disease Society of America. An official
ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am
J Respir Crit Care Med 2007;175:367-416.
48
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S4-1
A Sustainable Research and Development on Dokdo
Hyun Soo Rho
East Sea Research Institute (ESRI),
Korea Ocean Research and Development Institute (KORDI)
To further the dream of new, prosperous national marine system, the Dokdo Research Center has spearheaded
the protection and governance of the Dokdo territory through integrated research and systematic result
coordination based on the “Sustainable Use of Dokdo Act”.
By compiling marine data collected through scientific investigations on Dokdo and surrounding waters, the
program for “Sustainable Research and Development on Dokdo” has established efficient/sustainable use and
control measures for this island.
The data for Dokdo are being used to institute various strategies and a national policy for the environment of
the islands and neighboring seas, including strategies for control and preservation of the ecosystem, resource
securement, demarcation of the sea boundary between nearby countries, future spatial use, and strengthening of
the protection and governance of Dokdo.
The goals of this study were to characterize the topography and structural traits of Dokdo, conduct a field
survey and empirical analysis for ecosystem and environmental monitoring of the surrounding waters, and
coordinate data for Dokdo through the creation and operation of an integrated Dokdo database and web-site
(www.dokdo.re.kr).
These research results are important not only for the sustainable management and future use of this island but
also for their protection and governance; they also serve to increase awareness of Dokdo both within and outside
Korea through theses and conference presentations.
In addition, for the systemic control and efficient use of a Dokdo archive, all data were compiled in a
standardized database containing both previously collected data and data from our research team. All data on
Dokdo are available to the public through the Dokdo website as a national authorized portal site. The website
offers an abundance of information about Dokdo through a variety of activities, such as animations allowing the
user to indirectly experience the marine survey, communication between users, and an informational
communication that can both receive and offer professional advice.
Because the East Sea Research Institute was built in Jukbyeon-myun, Uljin-Gun, Gyeongbuk, the closest city
to Dokdo on the Korean mainland, we believe that the “Sustainable Research and Development of Dokdo”
program will become widely known throughout Korea, thus fostering more comprehensive and systematic
research endeavours.
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And, diverse scientific research into this island has allowed ‘Dokdo’ and the ‘East Sea’ to become
internationally recognized, both at home and abroad. The interest and involvement of the Korean people will
ensure that a national policy concerning Dokdo and the “Sustainable Research and Development of Dokdo”
program will be legislated to help resolve the issues related to Dokdo that are important to Korea’s national
security and national developmental potential.
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S4-2
The Study of Species Composition and Molecular Plant Geography
in Dokdo Island
Jae-Hong Pak*, Woong Lee, Don-Hwa Lee, and Mi-Seon Kim
Department of biology, College of natural sciences, Kyungpook National University, Daegu 702-701
Dokdo island belongs to the administrative district of Ulleung-gun, Gyeongsangbuk-do, Korea. It is Korean
easternmost island, situated in the middle of the East Sea, at a latitude of N 37°14′26.8″ and a longitude of E
131° 52′ 10.4″. Location is 87.4 km away from the island of Ulleungdo in the East Sea, and 216.8 km from
Jukbyeon, Gyeongsangbuk-do Province, and 157.5km away from the Japanese island of Oki. It is composed of
two main islands, Seodo (“West island”: 88,639m2) and Dongdo (“East island”: 73,297m2), as well as 89 tiny
rocks and reefs (25,517m2). Dokdo island is an oceanic island that created in 2~4.5 million years ago. It is
Boreal Kingdom, Sino-Japanese region, Korea and South Japan, Ulleungdo province in floral region so that has
independent characteristics different from the Japanese islands and Russia.
The flora of vascular plants in Dokdo island were confirmed to be consisted of 31 families, 56 genera, 57
species, 1 subspecies, 4 varieties totaling 62 taxa growing spontaneously. Woody plants were 8 taxa and
herbaceous plants were 54 taxa. Fern was 1 taxa, dicotyledons were 45 taxa and monocotyledons were 16 taxa.
Based on The specific plant species for environmental assessment by the Ministry of the environment in the
republic of Korea, a total of 13 taxa were identified including Orobanche coerulescens Stephan for the floristic
degree V. The dispersion type of plant migration in Dokdo island was investigated by classifying
anemochore(39 taxa, 62.9%), zoochore (12 taxa, 19.4%), hydrochore (2 taxa, 3.2%), artificial means.
To research the molecular distributional pattern of Dokdo island’s plants, we used 2 endemic taxa of Sedum
takesimense Nakai and Lonicera insularis Nakai in Ulleungdo and Dokdo island. L. insularis, grows on the
seaside in Ulleungdo and Dokdo island, is deciduous shrub. It has 2 hypotheses about its origin: 1) Japanese
Lonicera morowii A.Gray, which arrived Ulleungdo island by way of Dokdo island, is a progenitor of L.
insularis in Dokdo island according to Ulleungdo & Dokdo island’s characteristics of historical geology. 2) L.
insularis in Dokdo island planted by Ulleungdo island’s individual. To improve these 2 hypothesis, we
investigated cpDNA non-coding region trnL-trnF, trnS-trnG, petN-psbM, psbM-trnD, matK gene of L.
insularis. As a result, 4 region of trnL-trnF, trnS-trnG, psbM-trnD, matK gene corresponded with L. morowii’s
it completely but petN-psbM region has 2 different types. L. insularis has type 1 of Ulleungdo island, Dongdo of
Dokdo island, and type 2 of Ulleungdo island, Seodo of Dokdo island. L. morowii has only type 2. Accordingly,
this result supported both of 2 hypothesis. Because there were no records about L. insularis’s distiribution in
Dokdo island until L. insularis in Dokdo island planted by Ulleungdo island’s individual, the odds are against
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hypothesis 2.
S. takesimense is particularly found only in Ulleung and Dokdo islands. This is an endemic species first
reported by Nakai, that is the major species distributed in the Ulleungdo island. S. takesimense was classified
family Crassulaceae, genus Sedum, subgenus Aizoon, but different various opinions about their classification
were existed. So necessity of intensive studying was raised until the present. The comparative study of
Chloroplast DNA trnL/F intergenic region among the 32 materials in Ulleungdo and Dokdo island results 2
distinctive types of cpDNA haplotype. Type 1 showed in Albong region of Ulleungdo island and Dokdo island,
but Type 2 worked in Ulleungdo island only. Therefore, we got the result of two cpDNA lineages which has
separate molecular distributional pattern. This result suppose that S. takesimense originated from multiple
progenitors, also long-distance dispersal.
References
[1] Stuessy T.F. and Mikio Ono Evolution and speciation of island plants, 1-357, 1998.
[2] Pfosser M., Jakubowsky G., Schluter P.M., Fer T., Kato H., Stuessy T.F. and Sun B.Y. Pl. Syst. Evol., 256,
159-170, 2006.
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S4-3
Marine Invertebrate Fauna of Dokdo and Molecular
Phylogeography of Sea Slaters and Chitons in Korea and Japan
Ui Wook Hwang
Department of Biology, Teachers College & Institute for Phylogenomics and Evolution,
Kyungpook National University, Daegu 702-701
Benthic fauna were studied on the intertidal and subtidal rocky shores of Dokdo (island), Korea, between
August 2007 and June 2008. A total of 98 marine invertebrate species in 57 families was identified throughout
the study period, and 21 of those species are newly recorded including Porifera (n=1), Cnidaria (n=1),
Platyhelminthes (n=1), Mollusca (n=3), Annelida (n=4), Arthropoda (n=9), and Echinodermata (n=2). A
topographical description and ecological comment are given for each major species. Among the seven phyla
presented, Mollusca and Arthropoda predominated, accounting for >70-100% of the total taxa at each station. In
general, the faunal composition and distribution of invertebrates considered by habitat type (viz. attached,
sessile or mobile species) seemed to be closely related to the topographical characteristics of each site.
Altogether, the total of 403 species (172 families and 10 phyla) recorded in the present study and 13 earlier ones
indicate that the marine invertebrates inhabiting rocky bottoms in the Dokdo ecosystem show high and dynamic
biodiversity.
A sea slater Ligia exotica is a cosmopolitan species, widely distributed throughout all over the world. In
South Korea, it has been known there is only one species (L. exotica). Genetic diversity and phylogenetic
relationships among populations of L. exotica were examined based on the partial 16S rDNA (429bp) and
cytochrome C oxidase subunit I gene (COI, 539bp) of 310 individuals: 246 from various sites in South Korea
and 64 from Japan. Comparative analyses of the nucleotide sequences of 16S rDNA and COI revealed that L.
exotica consist of two distinct genetic lineages, A and B types. The sequence dissimilarity between the two
types is about 11.4 %. In total, 51 haplotypes (21 in A and 30 in B) were identified from the 310 individuals
examined in this study. In common, both the types A and B were found mixed in most collecting sites. However,
in the eastern coastline of the Korean Peninsula, the A type is more dominant, whereas the B type is more
dominant in in the western coastline vice versa. On the other hand, the results suggested that, in the four islands
(Ulleungdo, Dokdo, Jejudo and Tsushima islands), there exists only one type of the two exclusively and there
are shown absolute dominance of a type compared to the others. Through the AMOVA test, statistical
significance of genetic differences was examined between the two types of L. exotica and among individuals
within each of the two types. Consequently, the present data indicated that a large population of L. exotica in
South Korea and Japan consists of the two distinct mitochondrial genetic lineages.
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Liolophura japonica (Lischke, 1873) is one of the chitons distributed widely in the Korean Japanese coasts.
In this study, genetic diversity among Korean and Japanese L. japonica populations was analyzed, focusing on
those of Dokdo and Ulleungdo islands, Korea. Nucleotide sequence analyses of COI, 16S rDNA, and ITS
revealed that genetic diversity of L. japonica populations of Dokdo and Ullengdo islands was twice higher than
those of other examined regions. In total, 76 and 23 haplotypes were identified in COI and 16S rDNA,
respectively. Statistical analyses of the two mitochondrial genes showed that L. japonica comprises two
genetically distinct groups (types A and B). Type A has star-like structure indicative of a recent population
expansion, whereas type B has a more complicated genealogical pattern. The present study elucidated that type
A is widely distributed in the Korean and Japanese rocky coasts whereas type B is distributed below latitude of
35°N to 36°N. The two types of L. japonica are inhabitant below about 36°N with significant genetic difference
between them. In addition, phylogenetic analyses consistently showed that the two types were divided into two
separate clades. Comparative analyses of molecular, morphological, and ecological data of the two types A and
B strongly suggested that L. japonica may be divided into two independent species.
References
[1] Kim SH, and Kim YT Marine invertebrates II of Dokdo, Ministry of Environment, p. 32, 2006.
[2] Choe BL, Park JK and Lee JR Marine Molluscs of Ulrung and Dokdo Islands, Korean National Council for
Conservation of Nature, 353, 1996.
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S4-4
Exploring Microbial Functional Diversity of the Abyssal Seafloor in
the East Sea
Jong-Shik Kim
Research and Development Department, Gyeongbuk Institute for Marine Bioindustry (GIMB)
Marine microbial communities play roles in the functioning of abyssal seafloor ecosystems and are believed
to be major contributors to the biogeochemical cycles. Despite their importance, the vast majority of
microorganisms are uncultivated, thus their roles in the ecosystems are poorly understood. Recently, however,
the study of microbial diversity in the ocean has been advanced as presented by recent papers [1, 2, 3]. These
papers all provide invaluable methods and knowledge. Ocean samples, collected across Northwest Atlantic
through Eastern Pacific [1], at a depth of 4,000 m [2] and from ocean’s surface to near-sea floor depths [3], have
been studied for understanding microbial diversity. In order to understand microbial functional diversity of the
abyssal seafloor in East Sea, we are here investigating the microbial community organization and specific
functional adaptations from the level of the entire microbial communities to the individual isolates. Our goal is
to advance the understanding of structure-function relationships in microbial communities addressing the
followings: 1) diversity of nonculturable microorganisms in the abyssal seafloor in the East Sea, 2) diversity of
cultured microorganisms adapted to the deep-sea floor in the East Sea.
ACKNOWLEDGEMENTS
This work was supported by the Gyeongsanbuk-Do and Uljin-Gun’s research support program.
References
[1] Rusch D.B., A.L. Halpern, G. Sutton, K.B. Heidelberg, S. Williamson, S. Yooseph, D. Wu, J.A. Eisen, J.M.
Hoffman, K. Remington, K. Beeson, B. Tran, H. Smith, H. Baden-Tillson, C. Stewart, J. Thorpe, J.
Freeman, C. Andrews-Pfannkoch, J. E. Venter, K. Li, S. Kravitz, J. F. Heidelberg, T. Utterback, Y.-H.
Rogers, L.I. Falcon, V. Souza, G. Bonilla-Rosso, L.E. Eguiarte, D.M. Karl, S. Sathyendranath, T. Platt, E.
Bermingham, V. Gallardo, G. Tamayo-Castillo, M.R. Ferrari, R.L. Strausberg, K. Nealson, R. Friedman,
M. Frazier, and J.C. Venter. 2007. The Sorcerer II global ocean sampling expedition: northwest Atlantic
through eastern tropical Pacific. PLoS Biol. 5(3): e77.
[2] Konstantinidis K.T., J. Braff, D.M. Karl, and E.F. DeLong. 2009. Comparative metagenomic analysis of a
microbial community residing at a depth of 4,000 meters at station ALOHA in the North Pacific
subtropical gyre. Appl. Environ. Microbiol. 75(16): 5345-5355.
[3] DeLong E.F., C.M. Preston, T Mincer, V. Rich, S.J. Hallam, N.U. Frigaard, A. Martinez, M.B. Sullivan, R.
Edwards, B.R. Brito, S.W. Chisholm, and D.M. Karl. 2006. Community genomics among stratified
microbial assemblages in the ocean’s interior. Science 311: 496-502.
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S5-1
Practical Application of Environmental Microorganisms as
Bioresources
Sang Seob Lee
Dept. Biological Science, School of Natural Science, Kyonggi University
Environmental microorganisms can be used to help clean up pollution created by human activities, a process
called bioremediation. Various microorganisms can be used to consume spilled oil, solvents, pesticides, and
other environmentally toxic pollutants, The great diversity of microorganisms on Earth contains vast genetic
resources to mediate solutions for cleaning up the environment, and much research in this area is taking place at
present.
In this paper, recent progress in practical application of environmental microorganisms are briefly presented
with emphasis on their potential for bioresources.
Development of Biological Treatment System of Water Pollutants
The enhanced nutrients removal system for wastewater was developed using phototrophic purple non-sulfur
bacteria isolated from the streams in Kyonggi area, Korea using selective media, cultivated anaerobically under
the light (2,000 Lux). A 15-liter reactor modified from the A2/O process was employed for the cultivation and
the light apparatus was used for the dominant growth of photosynthetic bacteria. Experiments were performed
into two Phases and the results were compared: the synthetic wastewater was tested for the removal efficiency
of nutrients and organics during Phase 1 and the real wastewater during Phase 2. For both Phases, the F/M ratio
was maintained at 0.14-0.18 mg BOD5/mg MLVSS.day. Results showed that 97-99% of organics were removed
during Phase 1 and 96-99% during Phase 2. Nutrients (nitrogen and phosphorus) were also removed efficiently:
85-91% removal of T-N and 78-92% removal of T-P were achieved for Phase 1, and 76-89% removal of T-N
and 73-88% removal of T-P for Phase 2.
Development of Biodegradation System of Oil Contaminated Soil and Ground Water
In a oil contaminated site in In-chun, Korea, 43 strains of benzene degraders were isolated from soil by
growing on mineral medium with Benzene as a sole carbon source. Among them, 12 isolates grew up quickly.
12 strains were used for benzene removal screen test, and 7 strains for toluene removal were tested, respectively.
Consiquently, BJ10 which were identified as Pseudomonas sp. by 16S rDNA showed the highest removal
efficiency for benzene and toluene. In basal medium with 56 μmol benzene, BJ10 could degrade benzene over
95% and showed good biomass growth after 3 h of incubation(incubation condition : temperature; 30°C, cell
concentration; 1 g/L, pH8). With 47 μmol of toluene, BJ10 removed over 98% of toluene on liquid medium with
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same condition. Also, It can degrade toluene, ethylbenzene and o-Xylene without other nutrients.
Development of Biodegradation System of Explosives Contaminated Soil
Total 235 bacterial strains were screened for TNT (2.4.6-trinitrotoluene), 2,4 and 2,6-DNT (2,4 and
2,6-dinitrotoluene) removal, respectively. KT22 was identified as Serratia sp., KD4 was identified as
Pseudomonas sp., and KD6 was identified also as Pseudomonas sp., showed the highest removal efficiency for
TNT, 2,4-DNT, and 2,6-DNT, respectively.
Optimal conditions were shown cell concentration 1.0 g/L, pH 7.0, and temperature 25-30°C. The growth rate
was lower for 2,6-DNT, compared to TNT and 2,4-DNT. Mixed culture of KT22 and Bacillussp. showed
increased TNT removal efficiency.
In LB medium with 100 mg/L TNT, KT22 could degrade TNT over 97.7% and showed good biomass growth
after 6 h of incubation. The growth rate was lower for 2,6-DNT, compared to TNT and 2,4-DNT. KD4 and KD6
could degrade 2,4 /2,6-DNT over 99.0% and 92.66% after 12 h and 18 h of incubation, respectively. In Stanier’s
basal mineral medium, the growth rate(μ) of KT22 was 0.253 h-1, KD4 and KD6 were 0.212 h-1, 0.027 h-1.
Mixed culture of KT22 and Bacillus sp. showed increased TNT removal efficiency.
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S5-2
Current Status and Future Plan of KCTC
Jung-Sook Lee
Korean Collection for Type Cultures (KCTC), Biological Resource Center (BRC),
Korea Research Institute of Bioscience and Biotechnology (KRIBB),
111 Gwahangno, Yusong-gu, Daejeon 305-806
Tel: +82-42-860-4600, Fax: +82-42-860-4677, E-mail: [email protected]
The largest and oldest culture collection in Korea, Korean Collection for Type Cultures (KCTC) has three
functions as follows. 1) We carry out the collection and preservation of core biological resources from home and
abroad, offering public support by distributing biological resources to academia, industry, and research
institutions, and organizing patent strain deposit. Especially, we were designated as an International Depositary
Authority (IDA) under Budapest Treaty by World Intellectual Property Organization (WIPO). Now we can
manage about 17 kinds of biological resources including bacteria, yeast, fungi, algae, animal and plant viruses,
embryos, animal and plant cell cultures, seeds, RNA and etc. We manage more than 18,000 biological resources
including patent strains. Every year over 1,700 strains are newly added to KCTC. We also distribute more than
5,200 strains every year to home and abroad. 2) We develop the platform technology for the screening,
identification and preservation of useful biological resources. We publish more than 60 papers concerning
biological resources. Since 1997, we reported more than 160 new species. Last year, KCTC was cited by over
280 papers in PubMed search. 3) We are trying to construct on local and international network for biological
resources and related information, and support workshops, conferences, and consultation, etc.
As a national bio-infrastructure for biological resources, we perform the role of a biotechnology think-tank in
the field of bio R&D. KCTC plays an important part in networking among the collections in domestic, and
participates actively in international network among the collections in the world. We concentrate our efforts to
increase KCTC to international biological resource center.
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S5-3
Management of Microbial Genetic Resources
in Korean Agricultural Culture Collection (KACC)
In-Cheol Park*, Hang-Yeon Weon, Seung-Beom Hong, Soon-Ja Seok, Soo-Jin Kim, Yi-Seul Kim,
and Soon-Wo Kwon
Korean Agricultural Culture Collection (KACC), National Agrobiodiversity Center, National Academy of
Agricultural Science, Rural Development Administration, Suwon 441-853
Korean Agricultural Culture Collection (KACC) was established in 1995, in order to conserve microbial
diversity in agro-environment and food, providing related services to professional microbial research societies.
KACC classifies and preserves diverse Korean microbial resources including bacteria, actinomycetes, yeasts,
filamentous fungi and mushrooms. Moreover, in collaboration with Centraalbureau voor Schimmelcultures
(CBS), Deutsche Smmlung von Microorganismen und Zellkulturen GembH (DSMZ), NITE Biological
Resources Center, and other international collections, type strains from overseas were kept in KACC.
A total number of microbial strains registered in the KACC was up to 12,924 by end of the year, 2009. The
17,800 herbarium specimens of mushrooms are preserved in Herbarium Conservation Center of National
Academy of Agricultural Science (HCCN).
Registered microbial strains were preserved and maintained by various methods such as lyophilization,
cryopreservation by liquid nitrogen and ultra-low temperature storage below -80°C, water storage, mineral oil
storage and other methodologies according to their preservation properties. The registered strains were filed up
to database in the KACC homepage (http://www.genebank.go.kr) to facilitate the efficient utilization of
microbial resources and to make them easily accessible through internet. In 2009, the number of distributed
isolates scored up to 3,992 strains including 1,973 strains of bacteria and 2,019 strains of fungi. The distributed
strains were used in microbial research and development at laboratory of universities or institutes. It is estimated
that import substitution corresponds to eighty million won through distributions by KACC.
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S6-1
Molecular Signature of DC Subsets during
Acute vs. Chronic Virus Infection
Sang-Jun Ha*, Yun-Hee Jeong, Young-Ho Bahn, Joon-Seok Park, Chan-Hee Park,
and Hyo-Jin Park
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749
Today, chronic viral infections such as HIV, HBV, and HCV, became a major issue to threaten public health.
Chronically infected individuals not only act as a reservoir for viral spread, but also chronic infection in general
increases the risk of subsequent diseases and secondary infections with other pathogens. In the face of
continuously spreading viruses, there is an urgent need to develop vaccines that can be used therapeutically.
Many chronic viral infections are marked by pathogen persistence and a generalized immunosuppression. The
exact mechanisms by which this occurs are still unknown. The strategy comparing immune responses after
acute vs. chronic viral infection might be one of the ways to uncover the mechanism of immunosuppression
caused by chronic viral infection. We analyzed the function and phenotype of both T cells and dendritic cells
(DCs) after acute vs. chronic infection. By comparing antigen-specific acute and chronic T cells, we found that
co-inhibitory molecules including Programmed death 1 (PD-1) are highly expressed on T cells, suppress their
function, and finally leads to a progressive exhaustion. We also observed that conversion of DC subtypes
priming T cells (from lymphoid DCs to myeloid or plasmacytoid DCs) occurred during chronic infection but not
acute infection. Especially, myeloid DCs showed high level expression of co-inhibitory molecules and low level
expression of co-stimulatory molecules during chronic virus infection. Therefore, the strategy both blocking
inhibitory signals on T cells/DCs and targeting antigen to proper DCs might overcome immunosuppression and
subsequently enhance the clearance of persistent virus.
References
[1] Ha SJ, West EE, Araki K, Smith KA, and Ahmed R Immunological Reviews 223, 317 (2008).
[2] Ha SJ, Mueller SN, Wherry EJ, Barber DL, Aubert RD, Sharpe AH, Freeman GJ, and Ahmed R Journal of
Experimental Medicine 205, 543 (2008).
[3] Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, Kalia V, Subramaniam S, Blattman JN, Barber DL,
and Ahmed R Immunity 27, 670 (2007).
[4] Blattman JN, Wherry EJ, Ha SJ, van der Most RG, and Ahmed R Journal of Virology 83, 4386 (2009).
[5] Brooks DG, Ha SJ, Elsaesser H, Sharpe AH, Freeman GJ, and Oldstone MBA Proceedings of the National
Academy of Sciences USA 105, 20428 (2008).
[6] Li Q, Skinner PJ, Ha SJ, Duan L, Mattila TL, Hage A, White C, Barber DL, O’Mara L, Southern PJ, Reilly
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CS, Carlis JV, Miller CJ, Ahmed R, and Hasse AT Science 323, 1726 (2009).
[7] Slifka MK, Homann D, Tishon A, Pagarigan R, and Oldstone MBA Journal of Clinical Investigation 111,
805 (2003).
[8] Zuniga EI, Liou LY, Mack L, Mendoza M, and Oldstone MBA Cell Host & Microbe 4, 374 (2008).
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S6-2
Options for Control of Pandemic Influenza:
Active and Passive Immunization
Huan H. Nguyen
International Vaccine Institute, Seoul, Korea and Department of Microbiology,
University of Alabama at Birmingham, USA
Pandemic influenza poses a serious threat to global health and the world economy. Highly pathogenic avian
influenza A virus (HPAIV) of the H5N1 subtype that has emerged since 2004, resulted in more than 430 cases
of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% and remains a
global threat because of its continued transmission among domestic poultry and wild birds. Options for control
of pandemic influenza are vaccination in combination with novel immunization routes and therapeutic
anti-virals including virus-specific antibodies (Abs).
The current influenza vaccines designed to inducing antibody (Ab) responses against viral surface antigens
(i.e., hemagglutinin [HA] and neuraminidase [NA]) is limited by the ability of the virus to mutate these major
antigenic glycoproteins. Vaccines that target determinants conserved among influenza A viruses (IAV) to
generate broad protection against infection with different influenza A subtypes (i.e., heterosubtypic immunity
[HSI]) remain elusive. We have currently developed a recombinant adenovirus (Ad) vector co-encoding HA
(H5 subtype) and a conserved ectodomain of matrix protein 2 (M2e) (AdH5/M2e) for induction of protective
immunity to H5N1 and other subtypes. Another approach in influenza vaccine development includes new
generation of live-attenuated vaccine based on genetic deletion of the nonstructural NS1 protein of IAV. Since
NS1 enables the virus to disarm the host cell type 1 IFN defense system, mutation or deletion of the NS1 gene
leads to attenuation of the viruses and enhances host antiviral response. Therefore, NS1 deleted viruses
(DelNS1) can serve as a candidate live-attenuated influenza vaccine that provides better protection than
inactivated vaccines and could induce HSI to infection with different influenza virus A subtypes. Sub-lingual
immunization has been found to be a safe and effective route for induction of protective specific immune
responses in systemic and mucosal compartments including respiratory tract. We found that sublingual
immunization with either AdH5/M2e or DelNS1 induces broad protective immunity to H5 viruses and other
influenza virus A subtypes including H1N1.
Passive immunization (the transfer of specific immunoglobulins/Abs to a previously non-immune recipient
host) could offer an alternative strategy to prevent and treat influenza virus infection and an additional
therapeutic option to antiviral drugs that are limited by widespread drug resistance among influenza virus strains.
Even after targeted vaccines become available, passive immunization could still have prophylactic effects and
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provides an additional countermeasure against influenza. Attempts to develop monoclonal Abs (mAbs) have
been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader
specificity in order to provide full protection since mAbs are generally specific for single epitopes. We found
that H5N1-specific immunoglobulins (IgY) prepared from eggs laid by H5N1-vaccinated hens protect against
infections with HPAIV H5N1 and related H5N2 strains. When administered intranasally before or after lethal
infection, IgY prevent disease or significantly reduce viral replication resulting in complete recovery from the
disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated
H1N1 A/PR/8/34 as a model virus for current pandemic H1N1/09 and found that such H1N1-specific IgY
protect mice from lethal influenza virus infection.
These results underscore the usefulness of recombinant Ad vectors encoding surface glycoprotein (HA) and
conserved protein (M2e) and NS1 deleted viruses (DelNS1) as vaccine candidates for control of pre-pandemic
H5N1 and newly emerging subtypes. In addition, our data on antiviral efficacy of IgY provide a
proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the
control of influenza outbreaks, including the potential H5N1 and current H1N1 pandemic.
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S6-3
Chromatin Remodeling, HIV Reservoir, and Drug Development for
Eradication of Chronic HIV Infection
Kee-Jong Hong
Center for Infectious Diseases, Korea National Institute of Health
Chronic infection of HIV in CD4+ T cell reservoir is considered as a major barrier to eradicate the infected
viruses from AIDS patients. During chronic reservoir development after integration of HIV, chromatin
remodeling through acetylation and deacetylation may have a key role for latent replication of proviral DNA.
We established four new latently HIV-infected cell lines showing unique characteristics about latency; four
Clicj cells from jurkat T lymphocytes and three NCHA cells from A3.01 T lymphocytes. Those cell lines were
different from another already developed latent HIV cell lines J1.1 and ACH2 in basic characteristics; receptor
distribution, p24 induction and chemokine production. Our microarray experiments using latent cells showed
higher expression of histone deacetlyase 4 and H2B compared to parent T cells (A3.01 and Jurkat). p24
production by HIV replication in latent HIV cells was increased by treatment of the novel synthetic HDAC
inhibitors (CG05 and CG06), also the acetylation level of all latent cell lines was decreased. These two novel
HDAC inhibitors may support the new strategy to break HIV reservoir when used with HAART or other
inhibitors.
This kind of approach will provide us helpful information to develop new advanced drug candidate for HIV
treatment, also can support new strategy for virus eradication from HIV infected patients.
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S6-4
Automated HTS/HCS for Antivirals Using Visual HIV Full
Replication Assays
Peter Sommer
Cell Biology of Retroviruses Unit, Institut Pasteur Korea, Gyeonggi-do
E-mail: [email protected]
There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human
immunodeficiency virus (HIV) infection. However, new anti-HIV agents and treatment strategies are still
needed to confront the emergence of drug resistance and various adverse effects associated with long-term use
of antiretroviral therapy and the inability to cure infected individuals. We developed visual, HIV full replication
assays and implemented them in high-throughput compound (n=200.000) and genome-wide siRNA screens,
which allowed the identification of a few thousand novel small molecules with potent anti-retroviral activity and
a few hundred host factors required for HIV infection, respectively. The identified compounds and host factors
are opening unexplored avenues to novel antiviral drug and target discovery and validation, and should feed the
drug development pipeline in the near future.
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S6-5
Probiotics as an Immune Modulator - Mechanism of Action
and Therapeutic Applications
Ho-Keun Kwon1, Choong-Gu Lee1, Jae-Seon So1, Chang-Suk Chae1, Ji-Sun Hwang1,
Anupama Sahoo1, Jong Hee Nam2, Joon Haeng Rhee2, and Sin-Hyeog Im1,2*
1
Department of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712
2
Chonnam National University Medical School, Gwangju 501-749
Introduction
Gastrointestinal microorganisms affect host physiology through diverse mechanisms, including modulation
of the host immune system. Probiotics are nonpathogenic microorganisms that confer a number of beneficial
effects on the health of the host (1). Among them, species of Lactobacilli and Bifidobacteria are prominent
probiotics with anti-inflammatory properties (2). For example, administration of Lactobacillus casei suppresses
pro-inflammatory responses by increasing IL-10 levels (3, 4), while Lactobacillus acidophilus increases Th1
type cytokines (5). However, many questions remain unanswered, such as which probiotic strains are the most
effective in modulation of specific immune disorders and how orally administrated probiotics affects systemic
immune system (6).
In this study, we sought to identify which probiotics, or mixture of probiotics, could confer potent
anti-inflammatory effects by increasing CD4+Foxp3+ regulatory T cells (Tregs). The forkhead family protein
Foxp3 is a transcription factor highly expressed in CD4+ Tregs. It is a regulator of T cell tolerance, and is
necessary for the development and function of Tregs (7).
Methods
Following in vitro and in vivo functional studies on Treg generation, we developed a probiotics mixture that
exhibits potent anti-inflammatory properties and investigated its modulation of diverse immune disorders. The
probiotics mixture, designated IRT5, consists of a combination of five probiotic strains. The effect of IRT5
administration on host immune system was investigated.
Results and conclusion
Oral administration of IRT5 induced T and B cell hypo-responsiveness without inducing apoptosis. IRT5
administration increased the level of CD4+Foxp3+ Tregs in mesenteric lymph nodes (MLN) by augmenting
Foxp3+ levels in CD4+CD25- T cells. Conversion of T cells into Foxp3+ Tregs is directly mediated by regulatory
dendritic cells (rDCs) that express increased levels of IL-10, TGF-β, Cox2 and indoleamine 2,3-dioxygenase
(iDO). Administration of IRT5 suppressed the progression of experimental inflammatory bowel disease (IBD),
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atopic dermatitis (AD) and rheumatoid arthritis (RA). In addition, migration of Tregs to inflammatory regions in
response to chemokines (CCL1 and CCL22) and their receptors (CCR4 and CCR8) mediated disease
suppression. Our results present the first evidence of generation of CD4+CD25-Foxp3+ Tregs in response to
probiotics, an effect that may be therapeutically useful for the modulation of inflammatory immune disorders.
Acknowledgements
This research was supported by grants from the Regional Technology Innovation Program of the MOCIE
(RT105-01-01) and the BioGreen 21 Program (20070501034009; PJ007054) in Rural Development
Administration and an Agricultural R&D Promotion Center, Republic of Korea.
References
[1] Marteau P (2006) Gut 55(12):1692-1693.
[2] Braat H, et al. (2004) Am J Clin Nutr 80(6):1618-1625.
[3] So J-S, et al. (2008) Mol Immunol 46(1):172.
[4] So J-S, et al. (2008) Mol Immunol 45(9):2690.
[5] Sudo N (2002) Clinical & Experimental Allergy 32(7):1112-1116.
[6] Shanahan F (2003) Scand J Gastroenterol Supplement (237):34-36.
[7] Ziegler SF (2006) FOXP3 Annu Rev Immunol 24(1):209-226.
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S7-1
Adaptive Cellular Survival Response to Oxidative and
Inflammatory Stresses Induced by Helicobacter pylori
Young-Joon Surh
College of Pharmacy, Seoul National University, Seoul 151-742
Accumulating evidence from preclinical and clinical studies suggest that chronic infection and inflammation
contribute the carcinogenesis. Helicobacter pylori infection has been implicated in gastritis and peptic ulcer,
which can eventually progress to gastric cancer [1]. However, in a long-term study conducted in a wild-type
mouse model, H. pylori infection itself did not induce gastric cancer. To elucidate the mechanisms underlying
this discrepancy, we investigated the molecular mechanisms responsible for H. pylori-induced inflammatory
response in human gastric cancer (AGS) cells and genetically engineered animal models. Infection of AGS cells
with H. pylori up-regulated the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase,
enhanced secretion of interleukin-8, and increased phosphorylation of ERK. The nuclear translocation and
subsequent DNA binding of NF-kappaB, which is known to regulate pro-inflammatory gene expression, were
augmented after H. pylori treatment. The DNA binding of AP-1 and the expression of c-Jun and phospho-c-Jun
were also induced by H. pylori infection. A wide array of antioxidant enzymes and other cytoprotective proteins
constitute a fundamental cellular defense system against oxidative and proinflammatory insults. Treatment of
AGS cells with H. pylori resulted in elevated expression of heme oxygenase-1 (HO-1), a representative
stress-responsive enzyme, and its mRNA transcript. The immunofluorescence staining, using an
FITC-conjugated HO-1 antibody, verified the elevated cytoplasmic localization of HO-1 after H. pylori
treatment. The H. pylori treatment enhanced antioxidant response element (ARE) binding of Nrf2, a
redox-sensitive transcription factor responsible for regulating expression of many antioxidant and
cytoprotective genes, including ho-1. Chromatin-immunoprecipitation and ARE-luciferase reporter gene assays
revealed that H. pylori treatment induced Nrf2 binding to the ARE site and that the binding site is located at -0.5
kB of human ho-1 promoter region. After repeated H. pylori inoculation (x 3), mouse stomach tissues were
collected at 16 weeks. Following inoculation at 1, 4, 8 and 16th week of H. pylori inflammation was evident in
gastric mucosa, and there was the Nrf2 activation in mouse stomach as monitored by utilizing ARE transgenic
mice harbouring the human placenta alkaline phosphatase reporter gene. Analysis of stomach tissues collected
from Nrf-2-/-, Nrf-2+/- and Nrf-2+/+ mice infected with H. pylori for 16 weeks revealed that H. pylori-induced
inflammation as well as oxidative stress was more pronounced in Nrf2 knock-out mice than in hetero and
wild-type mice. Taken together, H. pylori infection can trigger the proinflammatory response through
up-regulation of COX-2, which provokes adaptive cellular defense response through Nrf2-mediated
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upregulation of antioxidant enzyme, such as HO-1, thereby protecting cells and tissues against subsequent
prooxidative and proinflammatory insults. Some phytochemicals, by stumulating Nrf2-driven cytoprotective
gene expression, may exert chemoprotective and chemopreventive eddects against H. pylori-induced gastritis
and gastric carcinogenesis [2].
Figure 1. Role of Nrf2 in adaptive survival response to H. pylori-induced oxidative and inflammatory gastric damage.
Acknowledgements
This work was supported by the National Research Foundation (NRF) grant (20100001707) through the
Ministry of Education, Science and Technology (MEST), Republic of Korea
References
[1] M. Miyamoto, K. Haruma, M. Yoshihara, T. Hiyama, M. Sumioka, T. Nishisaka, S. Tanaka, and K.
Chayama Dig. Dis. Sci., 48, 9.68-975, 2003.
[2] Y.-J. Surh Nature Rev. Cancer, 3, 768-780, 2003.
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S7-2
Physiological Functionalities of Small Peptides
Toshiro Matsui, PhD
Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, 812-8581, Japan
Tel/Fax: +81-92-642-3012
E-mail address: [email protected]
Bioactive peptides derived from natural proteins by enzymatic hydrolysis or fermentation have been
examined for the years whether they can prevent life-style related diseases, including hypertension, diabetes,
atherosclerosis and renal dysfunction.
The well-recognized physiological functionality of small peptides is a preventive potential against elevated
blood pressure in mild hypertensive subjects. For the effect, much interest has been focused on their inhibitory
potential of angiotensin I-converting enzyme (ACE) that is closely associated with the production of pressor
hormone, angiotensin (Ang) II from Ang I or the onset of hypertension. More than 400 ACE inhibitory peptides
have been reported so far. It was evidently proven that some ACE inhibitory peptides had a significant blood
pressure (BP) lowering effect in mild hypertensive subjects. A good example is a di-peptide-induced
anti-hypertensive effect, in which Val-Tyr isolated from sardine muscle hydrolysate showed a ca. 10 mmHg BP
reduction in human study for a 1-month protocol. Val-Tyr also claimed another physiological potential, since it
suppressed tissue renin-angiotensin systems in aorta and kidney, not in blood system in transgenic
Tsukuba-hypertensive mice bearing the human renin-angiotensin system.
To clarify the effect of small peptides on vessel function(s), ex vivo vascular contractive experiments were
conducted in a rat aorta-Magnus force measurement. As a result, we obtained an interesting finding that Val-Tyr
evoked vascular relaxation on thoracic aorta from SHR in endothelium- and ACE inhibition-independent
manners, although it has already been reported that longer or oligo-peptides such as Arg-Ala-Asp-His-Pro
(-Phe) could relax contractive tension of aorta in an endothelium-dependent manner through a promotion of
NO/cGMP vasorelaxation pathways. It indicates that small peptides may play a role in the regulation of vascular
response in vascular smooth muscle layer by alternative vasorelaxation mechanism(s). In a screening study of
vasoactive small peptides, basic amino acid-containing peptides such as Trp-His (EC50: 3.4 mmol/L) and
His-Arg-Trp (EC50: 1.2 mmol/L) were found to preferably relax the contractive aorta in an endotheliumindependent manner.
In vascular smooth muscle cell (VSMC) experiments both peptides showed unique anti-proliferative action,
in which they inhibited a WST-8 incorporation in Ang II-stimulated VSMCs, and a non-selective AT-related
receptors’ antagonist did not alter the effect. Moreover, elevated incorporation by Bay K8644 stimulation was
also inhibited by small peptides, which suggested that they might be associated with Ca2+ influx control. To
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make clear the possibility, further contractive experiments of small peptides in rat aorta were performed in
combination with L-type Ca2+ channel blockers. Although nifedipine, a dihydropyridine (DHP) type Ca2+
channel blocker, did not affect the Trp-His-induced relaxation; Verapamil, a phenylalkyl amine (PAA) type
Ca2+ channel blocker, reduced its relaxation power. It suggested that Trp-His as well as His-Arg-Trp would be
partly involved in the regulation of intracellular Ca2+ concentration ([Ca2+]i) in VSMC via an L-type Ca2+
channel. In a direct fluorescence measurement of [Ca2+]i by Fura-2 they significantly reduced the increase in
[Ca2+]i by Bay K 8644 stimulation to VSMC, suggesting their binding to the channel protein. On the other hand,
it was noted that no reduction of [Ca2+]i by their corresponding amino acids was observed, provided useful
information that any peptide skeleton must be essential for exerting vascular relaxation via [Ca2+]i suppression.
A marked reduction of [Ca2+]i by both vasoactive peptides in Ang II stimulation-VSMC study compared with
Bay K 8644 also revealed their alternative action toward [Ca2+]i regulation systems.
Our interest on peptide functionality has been then moved to preventive effect on vascular diseases, because
of their potent vascular relaxation peptides via inhibition of [Ca2+]i influx. Atherosclerosis is one of typical
vascular diseases related to VSMC migration or proliferation. In our challenging animal study using apo
E-deficient mice a 9-week-successive administration of Trp-His at a dose of 10 or 100 mg/kg/day resulted in a
significant reduction of atherosclerotic lesion area on aortic tree, while no difference in growth parameters and
lipid profiles was observed. The first finding that vasoactive di-peptide could ameliorate atherosclerosis without
any change in lipid profile may provide us a new physiological potential of small peptides.
Much interest has been focused on the elucidation of peptide functionalities. Recent studies by other
researchers also demonstrated some new findings regarding the improvement of renal dysfunction via
Ca2+-ATPase activation by Val-Tyr in renal proximal cells and hyperglycemia via post-transcriptional
down-regulation of SGLT1 expression by Gln-Cys-Pro or Gln-Ser-Pro. Given these diverse functionalities of
peptides, the intake of bioactive small peptides would be of great benefit for maintaining our homeostasis.
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S7-3
Anti-inflammatory Compounds from Medicinal Plants
Jae-Ha Ryu
College of Pharmacy, Sookmyung Women’s University, Seoul 140-742
Natural products have served as an important source of drugs since ancient times and about half of the useful
drugs today are derived from natural products. Contributing to this world-wide attention towards formulations
based on natural products are their low or absent toxicity, their complete biodegradability, their availability
from renewable sources, and, in most cases, their low-cost if compared with those of compounds obtained by
total chemical synthesis. Chemodiversity in nature, e.g. in plants, microorganisms and marine organisms, still
offer a valuable source for novel lead discovery for the drug development. In fact, individual plant species may
contain over one thousand chemical substances and only a minor fraction of 300,000 plant species has been
studied for medical application. But rapid identification of the bioactive compounds of natural product mixtures
remains critical factor to ensure that this tool of drug discovery can compete with recent developed techniques
such as chemical compound libraries and high-throughput screening of combinatorial synthetic efforts.
Recently, owing to the renewed attention to pharmaceuticals, agrochemicals and nutraceuticals obtained from
natural sources, the study of bioactive secondary metabolites, traditionally carried out by chemists, has
increasingly attracted the attention of pharmacologists, biologists, botanists, agronomists, etc., stimulating
cooperative work. So, we need to reinforce the interdisciplinary approach to the study of bioactive natural
products for the successful drug development.
In order to find new ant-inflammatory compounds from medicinal plants, we have screened bioactive
compounds by using the activated macrophage culture system. Activated macrophages produce proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1 and 6, and nitric oxide (NO).
The overproduction of NO by the oxidation of L-arginine by inducible nitric oxide synthase (i-NOS) activates
the cyclooxygenase-2 (COX-2) resulting in markedly increased release of pro-inflammatory prostaglandins
(PGs). In the presence of superoxide anion (O2-) NO can be converted into peroxynitrite (ONOO-) that is a
highly reactive molecule capable of oxidizing proteins, lipids, and DNA.
We have purified compounds that inhibited the production of NO in LPS-activated macrophages by
activity-guided purification, and indentified their structures by using spectroscopic methods. We identified
sesquiterpenes, polyacetylenes, diarylheptanoids, butanolides, flavonoids, lignans as active compounds from
Allium sativum, Alpinia officinarum, Angelica gigas, Artemisia iwayomogi, Atremisia princeps, Broussonetia
kazinoki, Carpesium macrocephalum, Curcuma zedoaria, Machilus thunbergii, Magnolia fargesii, Magnolia
obovata, Opuntia ficus, Panax ginseng, Perilla frutescens, Psoralea corylifolia, Saussurea lappa, Siegesbeckia
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glabrescens, Torilis japonica, Tussilago farfara, Xanthium strumarium. They showed their activity through the
suppression of i-NOS/COX-2 expression via the modulation of NF-κB activity and I-κB degradation and also
through the scavenging of peroxynitrite. I will discuss the purification, structural determination, action
mechanism of active principles from medicinal plants.
Fig. 1. Chemical structures of compounds 1-4 (A) and HMBC correlations of 4 from Magnolia fargesii (B). The effects of
4 on I-κB degradation, nuclear translocation of p65 (C), production of PGE2 (D) and expression of i-NOS and COX-2
(C), (D) in LPS-activated macrophages.
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S7-4
Extraction of Bioactive Materials from Rice Hulls
Seung-Cheol Lee
Department of Food Science and Biotechnology, Kyungnam University
Rice is the principal cereal in Asia, some countries in Africa, and Latin America. More than one million tons
of rice hulls (RHs) have been produced annually in South Korea after processing of rice. However, RHs are
wasted or destined to undervalued uses. Currently, agricultural and industrial residues are attractive sources of
natural antioxidants. The extraction of antioxidant compounds from residue materials such as hulls, seed coats,
peels, grape seeds, olive rape and cocoa byproduct has been reported. In general, seed coat plays an important
role in protecting seeds from oxidative damage because seed coat possesses large quantity of endogenous
antioxidant such as phenolic compounds.
Far-infrared (FIR) rays are defined as electromagnetic waves having a wavelength of longer than 4 μm but
shorter than microwave (λ>0.1 cm). FIR rays are biologically active and transfer heat to the center of materials
evenly without degrading the constituent molecules of surface. FIR, however, may have capability to cleave
covalent bonds and liberate antioxidants such as flavonoids, carotene, tannin, ascorbate, flavoprotein or
polyphenols from repeating polymers. After irradiation of far-infrared (FIR) onto RHs, methanolic extract was
prepared for the determination of antioxidant ability. After 30 min of FIR treatment, the radical scavenging
activity (RSA) and total phenol contents (TPC) of RH extracts increased from 47.74% to 79.63% and from 0.12
mM to 0.19 mM, respectively, compared to control. The inhibition of lipid peroxidation in extracts was also
increased from 41.07% to 47.96%. According to the GC-MS analysis, more phenolic compounds (p-coumaric
acid, 3-vinyl-1-oxy benzene, p-hydroxy benzaldehyde, vanillin, p-hydroxy benzoic acid, and 4,7-dihydroxy
vanillic acid) were detected in FIR-irradiated RH extract. These results indicated that FIR radiation onto RH
could liberate and activate covalently bound phenolic compounds that have antioxidant activities.
Subcritical water (SCW) is hot water under pressure sufficient to maintain water in the liquid state.
Antioxidant characteristics of SCW extracts from RHs was also evaluated. The TPC in SCW extracts of RHs
significantly increased with increasing temperature and treatment period. The TPC of RHs extract increased
from 165.1 μM of extraction at 25°C for 10 min to 270.2 μM of 200°C for 60 min. Antioxidant activity was
evaluated by determining RSA. The RSA of SCW extracts of RHs was also significantly increased with
increasing temperature and treatment period. The RSA of RH extract increased from 8.40% of extraction at
25°C for 10 min to 79.33% of 200°C for 60 min. The effect of SCW extraction temperatures and treatment
periods on TPC and RSA showed almost same trends.
The cytotoxic and antitumor activity of methanolic extract of rice hulls (MERH) were also evaluated by the
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MTT-dye reduction assay against human colon cancer cells, and the colonic aberrant crypt foci (ACF) assay in
1,2-dimethylhydrazine (DMH)-injected F344 male rats, respectively. MERH was found to be highly cytotoxic,
with IC50 values of 0.5 μg/ml in vitro. Forty weeks of MERH supplementation (50 mg/kg body weight/day)
reduced colonic pre-neoplastic ACF formation by 35% (p<0.01). An active compound, momilactone B, was
isolated from MERH by silica gel chromatography, Sephadex LH-20 chromatography, and HPLC. The
cytotoxic activity of momilactone B was evaluated by the MTT-dye reduction, lactate dehydrogenase (LDH)
and colony-forming ability assays in human colon cancer HT-29 and SW620 cells.
The results indicated that FIR irradiation or SCW extraction could be an excellent method for recovering
antioxidant materials from RHs, and momilactone B from RHs might be a new candidate for chemotherapeutic
agent against human colon cancer.
References
[1] Lee, S.C., Kim, J.H., Jeong, S.M., Kim, D.R., Ha, J.U., Nam, K.C., and Ahn, D.U. Journal of Agriculture
and Food Chemistry, 51, 4400, 2003.
[2] Kim, S.J., Park, E., Park, H.R., and Lee, S.C. Journal of Agriculture and Food Chemistry, 55, 1702, 2007.
[3] Park, S.M. and Lee, S.C. Food Science and Biotechnology, 18, 1435, 2009.
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S8-1
Differential Roles of Yeast Glutamate Dehydrogenase Isozymes in
Maintaining Resistance to Stress-Induced Apoptosis
Pil Jae Maeng*, Kyung Jin Kim, and Yong Joo Lee
Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 305-764
Glutamate is synthesized by glutamate dehydrogenases (GDHs). The yeast Saccharomyces cerevisiae has
three isoforms of GDH encoded by three distinct genes, GDH1, GDH2, and GDH3. Gdh1 and Gdh3 catalyze
the NADPH-dependent synthesis of glutamate from α-ketoglutarate, while Gdh2 catalyzes NAD+-dependent
reconversion of glutamate to ammonia and 2-ketoglutarate.
Here, we report differential roles of the two NADPH-dependent GDHs in stress-induced apoptosis. Cells
with ∆gdh1 mutation exhibited significantly decreased survival rate when exposed to either heat or oxidative
stress in pre-stationary phase culture, however, showed little difference compared to wild-type cells in stationary
phase culture. On the other hand, ∆gdh3 cells showed dramatically increased sensitivity to heat and oxidative
stress in stationary culture compared to wild-type cells, but moderately higher sensitivity in pre-stationary
culture. The death of ∆gdh3 cells caused by oxidative stress was shown to be associated with typical apoptotic
hallmarks, i.e., ROS accumulation, nuclear fragmentation, DNA breakage, and phosphatidylserine translocation
[1, 2]. Upon long-term cultivation, ∆gdh3 cells showed increased potentials for both aging-induced apoptosis
and adaptive regrowth. In addition, ∆gdh3 cells showed higher tendency to glutathione (GSH) depletion and
subsequent ROS accumulation than the wild-type, which was rescued by exogenous GSH, glutamate, or
glutathione disulfide (GSSG). It is thus suggested that GSH deficiency in ∆gdh3 cells is caused by an
insufficient supply of glutamate necessary for biosynthesis of GSH rather than the depletion of reducing power
required for reduction of GSSG to GSH. We also found that deletion of GDH2 suppresses the stress-induced
apoptosis caused by the shortage of glutamate supply required for the biosynthesis of GSH.
Despite the insignificance of GDH1 for the resistance to stressed-induced apoptosis in stationary phase cells,
the level of GDH1 transcription remained consistently high throughout the 3-d culture period. However, the level
of Gdh1 protein sharply decreased in stationary phase cells. On the other hand, transcription of GDH3 was low
during pre-stationary phase, but significantly high during stationary phase. In addition, the level of Gdh3 protein
changed similarly. This result suggests that the differential roles of GDH1and GDH3 in maintaining resistance
to stress-induced apoptosis might be mediated by differential protein degradation, but not by their transcription.
References
[1] Lee, Y. J., K. L. Hoe, and P. J. Maeng. Mol. Biol. Cell 18, 3556 (2007).
[2] Madeo, F., E. Frohlich, and K. U. Frohlich. J. Cell Biol. 139, 729 (1997).
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S8-2
Cadmium Regulates Copper Homeostasis by Inhibiting Mac1
Activity, a Transcriptional Activator of Copper Regulon, in
Saccharomyces cerevisiae
Dong-Hyuk Heo, In-Joon Baek, and Cheol-Won Yun*
School of Life Sciences and Biotechnology, Korea University, Seoul
Physiological processes in virtually all organisms require metal ions as cofactors, and cellular depletion of
particular metals can cause defects in numerous biological functions. On the other hand, excessive
accumulation of certain metal ions induces the production of reactive oxygen species (ROS) and ultimately
leads to cell death [1, 2]. Living organisms rely on delicate mechanisms to maintain intracellular metal
homeostasis. Certain metal ions, such as cadmium, arsenate, lead, and mercury, are harmful to organisms. Thus,
cells have unique mechanisms to protect themselves from such deleterious metal ions, such as exporting them
from the cell [3]. Cadmium is among the better-known toxic metals that affect living organisms. Cadmium
toxicity has been studied using various model systems, ranging from bacteria to humans. The sources of
cadmium are numerous, and the diseases that are caused by cadmium are diverse and include conditions such as
gastroenteritis [4], Itai-Itai disease, osteoporosis [5], renal dysfunction [6], and bone pain [7]. Cadmium directly
damages the cell membrane and induces apoptosis [8], mitochondrial dysfunction [9], cancer [10, 11], and
ultimately cell death. Interestingly, cadmium cannot directly form ROS, but it can induce their formation by
inhibiting the scavenging activities of superoxide dismutase and catalase [11]. Furthermore, cadmium induces
mutations both directly by damaging DNA and indirectly by inhibiting the activity of repair systems [12],
although the mechanism of this inhibition is not completely understood.
The mechanism of cadmium toxicity has recently been studied at the molecular level, and a link to sulfur
metabolism was identified. Interestingly, sulfur compounds such as methionine, cysteine, glutathione, and their
complexes are involved in the detoxification of cadmium [13, 14]. For example, cDNA microarray data show
that cadmium up-regulates the expression levels of genes that are involved in sulfur metabolism in S. cerevisiae
[14]. In addition, cadmium inhibits the SCFMet30 pathway, preventing this pathway from degrading Met4 [13].
Met4 is a primary transcription factor for the genes that are involved in sulfur metabolism. When cells are
exposed to cadmium, Met4 is activated, and it up-regulates the biosynthesis of molecules that are involved in
sulfur metabolism, such as glutathione. These molecules then scavenge the cadmium. These results suggest that
sulfur metabolism is a key detoxification pathway in S. cerevisiae.
Another aspect of cadmium toxicity comes from the metal ion’s ability to arrest the cell cycle, especially
through the Rad53 pathway [15]. Cadmium activates the Mec1/Rad53 pathway and ribonucleotide reductase,
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increases the copy number of mtDNA, and ultimately induces cell death.
Cadmium competes with physiologically important metal ions in the uptake pathway, and this competition
has lethal effects on organisms. This process is one of the principal mechanisms of cadmium toxicity.
Furthermore, the P-type ATPase Pca1 pumps Cd2+ out of the cell and into the extracellular environment, and
this protein thereby contributes to Cd2+ tolerance [16].
In S. cerevisiae, the intracellular copper concentration is strictly regulated because copper is an essential
metal for enzyme activity [17], defense against oxidative stress [18], and the electron transport system [19, 20].
Copper is taken up from the environment by two copper transporters, Ctr1 and Ctr3, which are localized to the
plasma membrane [21, 22] and regulated by copper sensing transcription factor, Mac1 [21]. Ctr1 and Ctr3
confer a high-affinity copper binding activity. Another copper transporter, Ctr2, is localized to the vacuole
membrane and confers a low-affinity copper binding activity [23]. Interestingly, CTR3 is not transcribed in
laboratory S. cerevisiae strains, due to the insertion of transposable elements in the promoter region [23]. Thus,
Ctr1 is the only high-affinity copper transporter protein in S. cerevisiae. The copper taken up by transporters is
delivered to target organelles by intracellular delivery systems, such as Atx1, Ccs, and Cox17 [17-20]. These
proteins deliver copper to the Golgi, Sod1, and the mitochondria, respectively.
The Mac1 protein [21] is a transcriptional activator that regulates the expression of the copper metabolism
genes mentioned above. High concentrations of copper bind to Mac1 and then inhibit the DNA-binding activity
of Mac1 [24], thereby inhibiting transcription of its target genes. Multiple cysteine-rich domains are found in
the C-terminus of the Mac1 protein [25], and these domains may confer copper-binding activity.
Interestingly, copper metabolism is regulated in a concerted manner with iron metabolism[26]. For example,
copper can affect iron metabolism through the regulation of the ferroxidase Fet3 protein. The Fet3/Ftr1 complex
comprises the reductive iron uptake system of S. cerevisiae, in which the ferrous form of iron is oxidized by
surface ferroxidase Fet3 and then taken up by iron permease Ftr1. The enzymatic activity of Fet3/Ftr1 depends
largely upon cellular copper utilization.
In this paper, we have identified a novel mechanism of cadmium toxicity in which cadmium inhibits Mac1
activity and, therefore, reduces CTR1 transcription. In addition, cadmium interferes with iron use by inhibiting
Fet3 and this leads to changes in iron and copper homeostasis in S. cerevisiae.
References
[1] Halliwell, B. and Gutteridge, J. M. (1984) Oxygen toxicity, oxygen radicals, transition metals and disease.
Biochem J. 219, 1-14.
[2] Lefebvre, V. and Buc-Calderon, P. (1995) Desferal prevents against cell lysis induced by hydrogen
peroxide to hypoxic hepatocytes: a role for free iron in hypoxia-mediated cellular injury. Chem Biol
Interact. 94, 37-48.
[3] Kim, D. Y., Bovet, L., Maeshima, M., Martinoia, E. and Lee, Y. (2007) The ABC transporter AtPDR8 is a
cadmium extrusion pump conferring heavy metal resistance. Plant J. 50, 207-218.
[4] Andersen, O., Nielsen, J. B. and Svendsen, P. (1988) Oral cadmium chloride intoxication in mice: effects
of dose on tissue damage, intestinal absorption and relative organ distribution. Toxicology. 48, 225-236.
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[5] Bhattacharyya, M. H., Whelton, B. D., Stern, P. H. and Peterson, D. P. (1988) Cadmium accelerates bone
loss in ovariectomized mice and fetal rat limb bones in culture. Proc Natl Acad Sci U S A. 85, 8761-8765.
[6] Itokawa, Y., Nishino, K., Takashima, M., Nakata, T., Kaito, H., Okamoto, E., Daijo, K. and Kawamura, J.
(1978) Renal and skeletal lesions in experimental cadmium poisoning of rats. Histology and renal function.
Environ Res. 15, 206-217.
[7] Wilson, A. K. and Bhattacharyya, M. H. (1997) Effects of cadmium on bone: an in vivo model for the early
response. Toxicol Appl Pharmacol. 145, 68-73.
[8] Coonse, K. G., Coonts, A. J., Morrison, E. V. and Heggland, S. J. (2007) Cadmium induces apoptosis in the
human osteoblast-like cell line Saos-2. J Toxicol Environ Health A. 70, 575-581.
[9] Muller, L. (1986) Consequences of cadmium toxicity in rat hepatocytes: mitochondrial dysfunction and
lipid peroxidation. Toxicology. 40, 285-295.
[10] Gunn, S. A., Gould, T. C. and Anderson, W. A. (1963) Cadmium-Induced Interstitial Cell Tumors in Rats
and Mice and Their Prevention by Zinc. J Natl Cancer Inst. 31, 745-759.
[11] Huang, Y. H., Shih, C. M., Huang, C. J., Lin, C. M., Chou, C. M., Tsai, M. L., Liu, T. P., Chiu, J. F. and
Chen, C. T. (2006) Effects of cadmium on structure and enzymatic activity of Cu,Zn-SOD and oxidative
status in neural cells. J Cell Biochem. 98, 577-589.
[12] Potts, R. J., Bespalov, I. A., Wallace, S. S., Melamede, R. J. and Hart, B. A. (2001) Inhibition of oxidative
DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein.
Toxicology. 161, 25-38.
[13] Barbey, R., Baudouin-Cornu, P., Lee, T. A., Rouillon, A., Zarzov, P., Tyers, M. and Thomas, D. (2005)
Inducible dissociation of SCF(Met30) ubiquitin ligase mediates a rapid transcriptional response to
cadmium. Embo J. 24, 521-532.
[14] Momose, Y. and Iwahashi, H. (2001) Bioassay of cadmium using a DNA microarray: genome-wide
expression patterns of Saccharomyces cerevisiae response to cadmium. Environ Toxicol Chem. 20,
2353-2360.
[15] Yen, J. L., Su, N. Y. and Kaiser, P. (2005) The yeast ubiquitin ligase SCFMet30 regulates heavy metal
response. Mol Biol Cell. 16, 1872-1882.
[16] Adle, D. J., Sinani, D., Kim, H. and Lee, J. (2007) A cadmium-transporting P1B-type ATPase in yeast
Saccharomyces cerevisiae. J Biol Chem. 282, 947-955.
[17] Lin, S. J. and Culotta, V. C. (1995) The ATX1 gene of Saccharomyces cerevisiae encodes a small metal
homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Acad Sci U S A. 92,
3784-3788.
[18] Culotta, V. C., Klomp, L. W., Strain, J., Casareno, R. L., Krems, B. and Gitlin, J. D. (1997) The copper
chaperone for superoxide dismutase. J Biol Chem. 272, 23469-23472.
[19] Beers, J., Glerum, D. M. and Tzagoloff, A. (1997) Purification, characterization, and localization of yeast
Cox17p, a mitochondrial copper shuttle. J Biol Chem. 272, 33191-33196.
[20] Glerum, D. M., Shtanko, A. and Tzagoloff, A. (1996) Characterization of COX17, a yeast gene involved in
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copper metabolism and assembly of cytochrome oxidase. J Biol Chem. 271, 14504-14509.
[21] Dancis, A., Haile, D., Yuan, D. S. and Klausner, R. D. (1994) The Saccharomyces cerevisiae copper
transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in
copper uptake. J Biol Chem. 269, 25660-25667.
[22] Knight, S. A., Labbe, S., Kwon, L. F., Kosman, D. J. and Thiele, D. J. (1996) A widespread transposable
element masks expression of a yeast copper transport gene. Genes Dev. 10, 1917-1929.
[23] Rees, E. M., Lee, J. and Thiele, D. J. (2004) Mobilization of intracellular copper stores by the ctr2 vacuolar
copper transporter. J Biol Chem. 279, 54221-54229.
[24] Keller, G., Bird, A. and Winge, D. R. (2005) Independent metalloregulation of Ace1 and Mac1 in
Saccharomyces cerevisiae. Eukaryot Cell. 4, 1863-1871.
[25] Keller, G., Gross, C., Kelleher, M. and Winge, D. R. (2000) Functional independence of the two
cysteine-rich activation domains in the yeast Mac1 transcription factor. J Biol Chem. 275, 29193-29199.
[26] Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. and Klausner, R. D.
(1994) Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for
copper in iron transport. Cell. 76, 393-402.
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S8-3
Membrane Potential Changes by Proton Pumping Microbial
Rhodopsins
Kwang-Hwan Jung
Department of Life Science and Institute for Biological Interface, Sogang University, Seoul 121-742
([email protected])
Microbial rhodopsins are retinal-binding proteins which function as ion pumps and photosensory receptors.
A variety of rhodopsins was discovered in many habitats since proteorhodopsin, which functions as a
light-driven proton pump, was found in uncultured marine bacteria of the SAR86 group. Recently, it was
revealed that PR-like proteins exist in genomes of flavobacteria. It has been characterized the photochemical
features of rhodopsins which were isolated from Korea East Sea and the surface of North & South pole. All of
them have the proton acceptor (D85 in BR) and donor (D96 in BR) which mediate proton translocation from
intracellular region to extracellular region when it receives light energy, and their actual proton pumping
activity in vitro has been demonstrated. Additionally, flavorhodopsin from IMCC 1997 cells is very unstable
compared to proteorhodopsin, which may be explained by the primary amino acid sequence differences. In
order to measure the stability of FR, we used the method of heat endurance at 70°C, and some FR mutants
showed that their endurance time against heat was increased. We showed that the Helix E is important for
stability of flavorhodopsin. We also applied several rhodopsins to various environmental conditions such as E.
coli membranes state, membrane gel state, DDM solubilized state, OG solubilized state, and DOPC
reconstituted state. According to the light-induced difference spectroscopy, rhodopsins solubilized in 0.02%
DDM clearly showed photointermediates like M, and O
states which respond to the different wavelengths,
respectively. The laser-induced difference spectra
ATP
synthase
showed the fast formation and decay rate of
photointermediates in the solubilized samples which
contain DDM. For the application, we developed a
bio-ATP-synthesis system using ATP synthase of E. coli
PR
as a biocatalyst and a microbial rhodopsin as light
energy converter. GR was overexpressed in E. coli and
made inverted membrane vesicle so that it could contain
both of GR and ATP synthase. Gloeobacter rhodopsin
(GR) produce a proton gradient which can be used in
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driving force of synthesizing ATP by ATP synthase in this vesicle.
(This work was supported by grant from the 21C Frontier Microbial Genomics and Application Program)
References
[1] Lee KA, Jung KH. 2010. ATP regeneration system using E. coli ATP synthase and Gloeobacter
rhodopsin and its stability. J. Nanosci. Nanotechno. Accepted.
[2] Miranda MRM, Choi AR, Shi L, Bezerra AG, Jung KH, Brown LS. 2009. Feb 18. The Photocycle and
Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin. Biophys J. 96(4):1471-1481.
[3] Jung JY, Choi AR, Lee YK, Lee HK, Jung KH. 2008. May 28. Spectroscopic and photochemical analysis
of proteorhodopsin variants from the surface of the Arctic Ocean. FEBS Letters-Biophysics
582(12):1679-1684.
[4] Brown LS, Jung KH. 2006. June. Bacteriorhodopsin-like proteins of eubacteria and fungi: extent of
conservation of the haloarchaeal proton-pumping mechanism. Photochem. Photobiol. Sci. 5(6):538-546.
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S8-4
Different Role of the DosS and DosT Histidine Kinases in
Mycobacterial Response to Hypoxic Conditions
Jin-Mok Lee, Kwang-Jin Park, Min-Ju Kim, and Jeong-Il Oh*
Department of Microbiology, Pusan National University, Busan 609-735
The DosS (DevS) and DosT histidine kinases in mycobacteria are involved in their hypoxic adaptation and
entry into the latent state. Although exhibiting high sequence similarities in their primary structures, DosS and
DosT differ in redox-sensing mechanism: DosS senses the environmental redox state, while DosT recognizes
the presence of O2 [1, 2].
The DevS histidine kinase of Mycobacterium smegmatis contains a tandem of the GAF domains (GAF-A and
GAF-B) in its N-terminal sensory domain. The DevS protein is a hemoprotein, and a b-type heme is coordinated
by its first GAF domain (GAF-A) with His-150 providing a proximal axial ligand to the heme [3]. The heme
iron of DevS was in the ferrous (Fe2+) state when purified and was resistant to autooxidation from ferrous to
ferric (Fe3+) state in the presence of O2. The binding of O2 to the deoxy-ferrous heme led to a decrease in DevS
autokinase activity, whereas NO-binding did not [3].
Phylogenetic analysis of the GAF-A domains of DevS homologues revealed that the primary structure of
GAF-A domain of M. smegmatis DevS is closely related to that of DosT in M. tuberculosis (MTB) rather than
DosS in MTB. In good agreement with the result of the phylogenetic analysis, DosT of MTB complemented the
DevS-phenotype of M. smegmatis DevS mutant strain as judged by the hypoxic induction of hspX, a gene
belonging to the Dev regulon, whereas DosS did not. We also found through this complement test that DosT and
DevS play a crucial role in the hypoxic induction of the Dev regulon during the transition phase from aerobic to
hypoxic conditions in contrast to DosS.
Multiple alignment of the amino acid sequences of GAF-A domains of DevS homologues showed that seven
amino acids are conserved differently between DosS and DosT subclades. The role of these amino acids in
different redox-sensing mechanism between DosS and DosT was investigated. It was demonstrated that five
amino acids (E87, D90, H97, L118, and T169 in DosS of MTB) might be responsible for the DosS- and
DosT-specific redox sensing mechanism.
The three-dimensional structure of the GAF-A domain of DosS revealed that it has a GAF-folding structure
with a b-type heme which is similar to the structure of the known PAS domain [4].
References
[1] Kumar A, Toeldo JC, Patel RP, Lancaster JR, and Steyn JC. Proc. Nat. Acad. Sci. USA 104, 11568, 2007.
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[2] Sousa EHS, Tuckerman JR, Gonzalez G, and Gilles-Gonzalez MA. Protein Sci. 16, 1, 2007.
[3] Lee JM, Cho HY, Cho HJ, Ko IJ, Park SW, Baik HS, Oh JH, Eom CY, Kim YM, and Oh JI. J. Bacteriol.
190, 6795, 2008.
[4] Cho HY, Cho HJ, Kim YM, Oh JI, and Kang BS. J. Biol. Chem. 284, 13057, 2009.
84
Symposia
S9-1
Synthetic 5′-Untraslated Regions for Fine-Tunable and Predictable
Gene Expression in Escherichia coli
Sang Woo Seo1, Jae-Seong Yang2, Inhae Kim3, Sanguk Kim2,3, and Gyoo Yeol Jung1,2*
1
Department of Chemical Engineering, 2School of Interdisciplinary Bioscience and Bioengineering, and
Division of Molecular and Life Science, Pohang University of Science and Technology, Gyeongbuk 790-784
3
A large number of natural and unnatural products from microorganisms such as biologically active
compounds and commodity chemicals have attracted huge attention in the various industries including
pharmaceutical and chemical industries. However, functional improvement of microorganisms meeting with
commercial need is limited by the biological constraints developed during natural evolutionary process.
“Metabolic Engineering” aims the purposeful redesign of the biological systems and requires the accurate
information of the cellular metabolic networks and proper tools for the reconstruction of the biological systems.
Numerous regulatory elements such as promoter libraries and RBS calculator can be applied for the modulation
of gene expression. However, without carefully considering the structural information of 5′-unstranslated
region (5′-UTR) sequence, it is insufficient to precisely design and modulate the gene expression level. To
address this issue, in this study, we randomized 5′-UTRs maintaining ribosome binding affinity using
superfolder GFP as a reporting system in Escherichia coli. A mathematical model was constructed by mapping
between the secondary structures of 5′-UTRs and the expression level of superfolder GFP. Examples using the
other genetic contexts will show the potentials of this model to predict the precise expression level based on the
structural information and consequently will provide a valuable tool for “Pathway Synthesis” for the production
of biofuels and commodity chemicals including butanol, hydrogen, and amino acids.
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S9-2
Rewriting Biology with Gene Synthesis
Duhee Bang
Department of Chemistry, Yonsei University, Seoul 120-749
Advances in the chemical synthesis of DNA have facilitated our ability to synthesize genes at unprecedented
error rates. These synthetic capabilities along with emerging genomic technologies are now making it possible
to tackle the challenge of constructing new biological systems to address major problems in biology, medicine
and energy. I will present efforts to scale up DNA synthesis to millions base pairs at an affordable cost [1-3]. A
resulting revolution in the DNA synthesis will enable us to test our information-driven hypothesis by
reconstructing DNA sequences in a highly controlled manner. The development of an ultra-low cost synthetic
genomics platform will generate unprecedented opportunities in an emerging field of Synthetic Biology.
References
[1] D Bang, and GM Church, Nature Methods, 2008, 5, 37-39.
[2] HB Kim, HJ Han, and D. Bang, submitted.
[3] J Tian, H Gong, N Sheng, X Zhou, E Gulari, X Gao, and G Church. Nature, 2004, 432, 1050-1054.
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S9-3
From Oligos to Organisms
Mikkel Algire
Synthetic Biology and Bioenergy Group, J. Craig Venter Institute
The Synthetic Biology group has been trying to develop a minimal cell both to understand the fundamentals
of biology and to build a new cell and organism with optimized functions. In order to construct a synthetic cell
we need to synthesize and assemble a whole genome and activate or ‘boot up’ this synthetic genome.
As a first step toward the construction of minimal synthetic cell and to demonstrate that a bacterial genome
could be activated, we completely replaced the genome of a bacterial cell with one from another species by
transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides subsp. capri
was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. These
cells that result from genome transplantation are identical to the M. mycoides donor strain.
We have also developed whole genome assembly techniques to allow us to have nucleotide level control over
the synthetic genome. Using these tools assembled and cloned the synthetic Mycoplasma genitalium JCVI-1.0
genome in the yeast Saccharomyces cerevisiae by recombination to produce a 592-kb circle. We further
extended this approach by assembling the synthetic genome from 25 overlapping fragments in a single step. The
use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and
natural fragments.
In order to produce a synthetic cell, the genome assembly and genome transplant steps must be combined.
Following the chemical synthesis, assembly, and cloning of a bacterial genome in yeast the genome must be
transferred from yeast to a receptive cytoplasm. To demonstrate the ability to transplant a cloned bacterial
genome from yeast we cloned a M. mycoides genome as a yeast centromeric plasmid and then transplanted it
into M. capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast
genetic systems and then transplanted to produce a new strain of M. mycoides.
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S9-4
Artificial Assembly of a Minimal Cell
Giovanni Murtas
Centro Enrico Fermi, Piazza del Viminale 1, 00184 - Rome - Italy
Synthetic Biology approaches can assemble and/or reconstruct cell parts in synthetic compartments. A
minimal cell as a model for early living cells can be artificially constructed in the laboratory resuming the main
properties of a basic cell living system, a synthetic cell compartment or liposome to host a minimal metabolism
based on protein synthesis, and a shell and core reproduction mechanism. Starting from the minimal cell
assembly approach it is becoming realistic to construct artificial cells, and deliver cell-like bioreactors to
synthesize pure proteins/enzymes or isolate single pathways. These artificial cell-like systems could perform
different tasks in antimicrobial drug development, drug delivery and diagnostic applications
With a bottom up approach we are aiming to construct a minimal cell using a minimal set of extant enzymes
and/or genes to create basic bio-mechanisms in simple lipid compartments know as liposomes, to develop a
cell-system alive that means implementing in a liposome compartment a basic metabolism, based on protein
synthesis, and a self-replication mechanism to insure cell reproduction.1,2 These should be the essential
bio-mechanisms for a cell system to be defined alive and to qualify for the lowest degree of complexity
compatible with cellular life.
A suitable cell-compartment to build a cell model in the lab is represented by the liposome, a closed spherical
bilayer, considered the most likely precursor of early living cells. In order to establish a basic mechanism of
protein synthesis for a minimal metabolism, a set of 36 pure enzymes with purified ribosomes, energy and
nutrients has been implemented in the cell compartment known as the PURESYSTEM (PS).3
A second and fundamental milestone to achieve in this model is represented by the property, present in any
cell or living system, known as the ability to self-reproduce itself. This is to be distinguished into
cell-compartment or vesicle self-reproduction and core or genome reproduction. On this purpose we can foresee
a system where enzymes for lipid membrane synthesis are introduced within liposomes and drive the catalysis
required for the boundary layer reproduction. A simple biochemical mechanism can be proposed and this is to
introduce into vesicles a Fatty Acid Synthetase (FAS) enzyme for fatty acid synthesis. The FAS enzyme with
fatty acid catalysis could induce vesicle growth and shell self-reproduction by fatty acid incorporation into the
lipid bilayer. In particular the FAS-B type I from Brevibacterium ammoniagenes has been already characterised
and expressed in the heterologous system E. coli.4 FAS-B catalyze mainly the synthesis of palmitate.
Preliminary experiments have already shown that the FAS-B enzyme, as pure enzyme, can control endogenous
lipid synthesis within liposomes proving lipid pattern change of membrane vesicles due to fatty acids synthesis
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and incorporation5.
For a complete and autonomous shell self-reproduction process the genetic components must be introduced
within the model system, therefore the FAS-B gene together with the PS should be encapsulated into vesicles.
This is a system that would sustain the FAS-B expression and Fatty acid catalysis allowing a shell selfreproduction, from mother to daughter vesicles, for a few generations although the system would be exhausted
soon after. The main problem is connected with the limited amount of any PS enzyme available within each
vesicle that would diminish dramatically in a few generations.
To overcome the limiting condition of this protocell a set of genes encoding the PS components such as all the
aminoacyl-tRNA synthetases and translation factors, will be integrated into vesicles. In addition the genes for
the ribosomal proteins and rRNA required to assemble minimal ribosome will be synthesised using the T7 RNA
polymerase and the PS. The tRNA genes will be transcribed by a T7 RNA polymerase to complete the genetic
composition of the PS. Again, although the PS genome insertion into vesicle will help to sustain the vesicle
reproduction process, this will not stop the limitation caused from death by dilution of all the elements included.
The real self reproduction of biological cells is based on the formation of identical copies, containing each time
the full apparatus. Therefore conditions are required under which the ribosomal system and all enzymes
assisting transcription-translation for protein synthesis self-replicate.
This is in principle possible by introducing some of the enzymes/genes responsible for DNA replication such
as the DNA polymerase III, a DNA primase, helicase, ligase and gyrase, plus a gene for single-stranded DNA
binding protein. At this stage, to keep the cell model within a frame of low complexity and remain in the realm
of minimal living, nutrients and energy such as molecules of ATP, amino acids, nucleotides and other low
molecular weight components will be provided and not synthesized autonomously by the system. Finally the cell
model system should be tested in the laboratory to prove that it is able to evolve in a changing environment. 1,2,6
Synthetic Biology is now offering new technologies and approaches for artificial cell assembly. Artificial
cells are now prospects for biotechnical and biomedical applications7. A recent work on a cell-like bioreactor
has provided interesting suggestions to build new bioreactors for protein/enzyme synthesis within lipid vesicles8.
The artificial cell assembly exercise may shed light on the origin of living cells9 but at the same time can provide
the basic tools and lay the foundation for a new generation of artificial cell bioreactor systems for biotechnology.
A new cell-like structure can be developed using a simple lipid vesicle compartment, as described for the
EGFP synthesis in liposome10, with the same or similar channel pore exchange system as introduced by
Noireaux et al., but replacing the E. coli extract for protein synthesis with the pure set of enzymes (PS) to control
protein synthesis. More in details the PS system together with the α-hemolysin gene and any gene of interest can
be easily entrapped into lipid compartments or liposomes with the option to add post-translational modification
components or membrane proteins to specialise this vesicle bioreactors for biotechnological applications. These
ready formed liposomes can be fed in water solution with a mix of nutrients and energy to sustain the internal
reactions for a few days.
This would allow a long lasting, high yield, synthesis and catalysis with an high degree of pure products and
in the absence of inhibitors. This system would be suitable for testing artificial genomes, any single and/or
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artificial metabolic pathway or new pharmaceutical molecule isolated from the whole and complex metabolic
contest. The same artificial cell-like structure could be assemble to combat chronic infection known as biofilms
by synthesizing and delivering bactericides in situ.11 A similar technology could be promising in medical
diagnostic where a liposome could entrap an artificial metabolism and convert relevant molecular compounds
associated or released from any pathology into detectable fluorescent signals.
In conclusion spin-off projects are possible from the artificial minimal cell assembly approach where a pure
and minimal metabolism is encapsulated within liposomes this would be a cell-like device without capability of
self- replicate, and therefore a non bio-hazardous system. These artificial cell-like structures could be
engineered for specific applications to allow testing new artificial metabolisms, most promising in
pharmacology, medical diagnostic and drug delivery.
References
[1] P.L. Luisi, F. Ferri, P. Stano, Approaches to semi-synthetic minimal cells: a review, Naturwissenschaften,
2006, 93, 1-13, Review.
[2] G. Murtas, Construction of a semi-synthetic minimal cell: a model for early living cells, Orig Life Evol.
Biosph., 2007, 37, 419-22.
[3] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Cell-freetranslation
reconstituted with purified components. Nat. Biotechnol., 2001, 19, 751-5.
[4] H.P. Stuible, G. Meurer, E. Schweizer, Heterologous expression and biochemical characterization of two
functionally different type I fatty acid synthases from Brevibacterium ammoniagenes, Eur. J. Biochem.
1997, 247, 268-73.
[5] G. Murtas, Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal
cell. Systems and Synthetic Biology (DOI: 10.1007/s11693-009-90481)
[6] P.L. Luisi, in The emergence of life, 2006, eds. Cambridge University Press
[7] A. Pohorille, D. Deamer, Artificial cells: prospects for biotechnology, Trends Biotech, 2002, 20, 123-8.
[8] V. Noireaux, A. Libchaber, A vesicle bioreactor as a step towards an artificial cell assembly, Proc. Natl.
Acad. Sci U.S.A., 2004, 101, 17669-176747
[9] J.W. Szostak, D.P. Bartel & P. L Luisi, Synthesizing life, Nature, 409, 387-390
[10] G. Murtas, Y. Kuruma, P. Bianchini, A. Diaspro, P.L. Luisi, Protein Synthesis in liposomes with a minimal
set of enzymes. Biochem. Biophys. Res. Commun. 2007, 363(1), 12-7
[11] M.N. Jones. Use of liposomes to deliver bactericides to bacterial biofilms, Methods Enzymol., 2005, 391,
211-28.
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S9-5
Toward the Bacterial Minimal Chromosome
Young-Chang Kim1,2,3*, Mun-Sik Shin 1, Kyung-Bae Min2, Woo-Yeong Jang2, Ji-Eun Yu2,
Hye-Jin Bae 2, Joo-Han Gwak2, and Soon-Gee Cheong3
1
Department of Synthetic Biology, 2Department of Microbiology, 3Biotechnology Research Institute,
Chungbuk National University, Cheongju, 361-763
The minimal genome is the smallest possible group of genes that would be sufficient to sustain a cellular life
form under the most favorable conditions imaginable [1]. There are two ways to synthesize a minimal genome,
which are top-down and bottom-up approaches. We are interested in the bottom-up approach, in which the
major hurdle is how to design the minimal genome. We don’t know the principle of the organization of the
genome. Moreover to verify the design experimentally, we should have to deal with some 200 or more essential
genes. So, we use the divide-and-conquer strategy to solve the problems in designing the minimal genome [2].
We made E. coli having two chromosomes. One is a normal chromosome and the other is a model minimal
chromosome, in which the replication and segregation processes and their regulation are occurred
independently. Studies on the minimal chromosome will provide us useful information about designing the
minimal genome.
References
[1] Wegrzyn, G. The minimal genome paradox. J Appl Genet. (42), 385-392, 2001.
[2] Pal, D., Venkova-Canova, T., Srivastava, P., and Chattoraj, D. K. Multipartite regulation of rctb, the
replication initiator gene of vibrio cholerae chromosome ii. J Bacteriol. (187), 7167-7175, 2005.
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S10-1
Novel Enzymes and Metabolic Pathways in
Hyperthermophilic Archaea
Haruyuki Atomi1*, Takaaki Sato1, Yuusuke Yokooji1, Ryo Takemasa1, Tamotsu Kanai1,
and Tadayuki Imanaka2
1
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
2
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu 525-8577, Japan
E-mail: [email protected]
Thermococcus kodakaraensis is a hyperthermophilic archaeon isolated from Kodakara Island, Kagoshima,
Japan. The organism is an obligate anaerobe and heterotroph, and displays an optimal growth temperature of 85°C.
We have previously determined the entire genome sequence of T. kodakaraensis, and have estimated the presence
of 2,306 genes. Assignment of gene function based on primary sequence analysis is possible for approximately
half of the genes, and this enables us to predict the presence or absence of particular metabolic pathways in this
archaeon. Intriguingly, in many cases, although biochemical data suggest otherwise, there are pathways that are
incomplete judging from the genome data. One of the main objectives of our research is to identify the enzymes
or pathways that functionally replace these missing enzymes. Classical biochemical methods, combined with a
comparative genomics approach, have proven to be effective [1-3]. Once identified, the function of the
protein/gene in vivo is confirmed genetically using the gene disruption system developed for T. kodakaraensis.
We will introduce several examples of novel metabolic enzymes and pathways discovered in T. kodakaraensis,
focusing on our results concerning pentose metabolism and the coenzyme A biosynthesis pathway.
We will also describe recent improvements in the gene manipulation system developed in T. kodakaraensis.
The system now allows, in addition to multiple gene disruption, gene insertion (reporter genes/heterologous
protein production), promoter exchange (over-expression), tag insertion (purification), and signal peptide
insertion (protein secretion). Using these gene manipulation techniques along with the complete genome
sequence data of this organism, we are now capable of utilizing T. kodakaraensis as host cells for protein
production and cell engineering.
References
[1] Targeted gene disruption as a tool for establishing gene function in hyperthermophilic archaea. H. Atomi
and T. Imanaka. Ch. 13, pp. 213-223, In Thermophiles, CRC Press, London, 2008.
[2] Archaeal type III RuBisCOs function in a pathway for AMP metabolism. T. Sato, H. Atomi and T.
Imanaka. Science 315, 1003-1006, 2007.
[3] Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis
in the Archaea. Y. Yokooji, H. Tomita, H. Atomi and T. Imanaka. J. Biol. Chem. 284, 28137-28145, 2009.
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S10-2
Global Proteome Analysis of Sulfur-Reducing Hyperthermophilic
Archaeon Thermococcus onnurineus NA1
Jong-Soon Choi*, Seung Il Kim, and Young-Ho Chung
Division of Life Science, Korea Basic Science Institute, Daejeon 305-333
Thermococcus onnurineus NA1 is a representative of sulfur-reducing hyperthermophilic archaeon,
Thermococcus sp. T. onnurineus NA1 was isolated from a deep-sea hydrothermal vent area. The strain requires
elemental sulfur as a terminal electron and organic compounds such as peptides, amino acids and sugar as
carbon source. This strain shows the optimum growth at 80°C and pH 8.5. Recently, the genome sequence of T.
onnurineus NA1 was completed, in which its genome retains the metabolic pathway not only for organotrophic
growth but also for carboxydotrophic growth [1]. In the present study, we attempted (i) to analyze global
proteome of T. onnurineus NA1 at organotrophic and carboxydotrophic culture condition and (ii) to identify
hyperthermostable proteins by the enrichment of in vitro heat treatment. Furthermore, (iii) the recombinant
target thermostable proteins were characterized for the biophysical properties such as enzyme activity, substrate
specificity, and cation requirement for growth.
For the proteomic profiling, T. onnurineus NA1 cells grown in each culture conditions (yeast-peptone-sulfur
medium for organotrophic growth; carbon monoxide as a sole carbon for carboxydotrophic growth) were
harvested and lysed by boiling. The soluble proteins were prepared by centrifugation and separated on 12%
SDS-PAGE. The gel was fractionated according to molecular size and the sliced gel bands were subjected to
in-gel trypsin digestion. The resulting peptides were subjected to LC-MSMS analysis with Mascot v2.2
database for protein identification and relative quantification. For studying thermostable proteins,
organotrophic growth medium-grown T. onnurineus NA1 cells were harvested and cytosolic proteins were
extracted by the previous method [2]. The cytosolic fraction containing 1 mg protein was exposed to heat
treatment for 10 min to 24 h at 60°C, 80°C, and 100°C. Denatured and aggregated proteins were removed by
centrifugation and the resultant soluble fraction was performed by gel-based and shotgun proteomic analyses.
In the proteomic profiling study, we identified and quantified the proteomes of T. onnurineus NA1 cultured at
these two different cultures by SDS-PAGE/FT-LC-MS analysis; 1,268 and 1,572 proteins were identified from
YPS- and CO-culture medium, respectively. A total of 1,612 proteins were identified from T. onnurineus NA1,
in which the identification rate corresponds to ~82% of genome-the top score among the archaeon proteome
data for our best knowledge. By the analysis of functional categories of identified proteome, the relative
percentage of proteins belonging to energy production in the group of metabolism was 2-fold increased in
CO-culture condition compared to that in YPS-culture one. In particular, hydrogenase gene clusters responsible
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for biohydrogen production were prevalently increased in the carboxydotrophic growth. The altered metabolism
is probably due to the feasible proteomes of T. onnurineus NA1 for high biohydrogen production. In the
proteomic analysis of heat-stable proteins, 13 and 149 proteins were identified as intact ones after heat treatment
at 100°C for 2 h by 2DE/MALDI and SDS-PAGE/LC-MS analysis, respectively. Representative thermostable
ezymes like deblocking aminopeptidase and fructose 1,6-bisphosphatase were over-expressed in E. coli system.
Thermostability of these proteins was confirmed by enzyme activities and in silico TargetStar analyses [3].
In conclusion, the global proteome analysis suggests that T. onnurineus NA1 can give a possibility of the
industrial application for the production of biohydrogen and thermostable enzymes.
References
[1] Lee HS, Kang SG, Bae SS, Lim JK, Cho Y, Kim YJ, Jeon JH, Cha SS, Kwon KK, Kim HT, Park CJ, Lee
HW, Kim SI, Chun J, Colwell RR, Kim SJ, Lee JH, J Bacteriol, 190, 7491, 2008.
[2] Kwon SO, Kang SG, Park SH, Kim YH, Choi JS, Kim SI, Extremophiles 13, 379, 2009.
[3] Kim HJ, Moon EJ, Moon S, Jung HJ, Yang YL, Park YH, Heo M, Cheon M, Chang I, Han DS, Int J
Modern Physics C 18, 1513, 2007.
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S10-3
Transcriptome Analysis on a Heat Shock Regulon in the
Hyperthermophilic Archaeon, Thermococcus kodakaraensis
Tamotsu Kanai1*, Shogo Takedomi1, Tomoyuki Kamashita1, Masahiro Nakanosho1,
Shinsuke Fujiwara2, Haruyuki Atomi1, and Tadayuki Imanaka3
1
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan,
2
Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University,
2-1 Gakuen, Sanda, Hyogo 669-1337, Japan,
3
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis (Topt = 85°C) contains a gene
(TK2291) encoding a putative transcriptional regulator orthologous to the Pyrococcus furiosus Phr (Pf-Phr).
Pf-Phr represses genes encoding the small heat shock protein, an AAA+ ATPase, and Phr itself under normal
growth temperatures [1,2]. To examine the physiological roles of the Phr ortholog (Tk-Phr) and its contribution
to heat shock response, its deletion mutant (KHR1) was constructed. Growth measurements indicated that the
KHR1 cells showed similar specific growth rates with those of the wild-type strain, but an increase in cell yield
in the stationary phase was observed. To determine the genes that were regulated by TK2291, a whole genome
microarray analysis was performed between KHR1 and wild-type cells grown at 80˚C. Transcript levels of more
than 20 genes were significantly higher in KHR1 cells than in wild-type cells. The majority of these genes
contained a sequence motif virtually identical to that of Pf-Phr in their 5’-flanking regions. The TK2291 regulon
includes genes encoding the small heat shock protein (TK1155), an AAA+ ATPase (TK1157), prefoldin
(TK1121-TK1122), ATPase of the RecA superfamily (TK1590), and Tip49 (TK1199). On the other hand, more
than half of the members in the regulon encode either conserved or hypothetical proteins, raising the possibility
that these proteins participate in as-yet unidentified processes of the heat shock response in T. kodakaraensis [3].
Genetic and biochemical analyses of Tk-Phr are also discussed.
References
[1] Vierke, G., Engelmann, A., Hebbeln, C. and Thomm, M. (2003) J. Biol. Chem. Vol. 278, p. 18-26 (2003).
[2] Liu, W., Vierke, G., Wenke, A. K., Thomm, M. and Ladenstein, R. J. Mol. Biol. Vol. 369, p. 474-488
(2007).
[3] Kanai, T., Takedomi, S., Fujiwara, S., Atomi, H. and Imanaka, T., J. Biochem., Vol. 143, p. 361-370
(2010).
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S10-4
Molecular Architecture and the Mechanics of Lon,
a Protease-Chaperone Machine
Sun-Shin Cha
Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan 426-744
The ATP-dependent Lon protease, which has orthologs distributed in all kingdoms of life [1-3], is essential in
bacteria and other microorganisms under stress conditions and is needed for survival of mammalian cells
subjected to oxidative damage. Lon consists of a molecular chaperone belonging to the AAA+ family and a
protease with a serine-lysine catalytic dyad encoded in tandem in a single polypeptide. Here, we report the 2.0 Å
resolution crystal structure of Lon from Thermococcus onnurineus NA1 [4] (TonLon). Six subunits of TonLon
assemble into a cylindrical structure with a sequestered internal chamber harboring the proteolytic active sites
accessible only through restricted axial channels. Alternating subunits exist in two different nucleotide states
displaying different domain orientations and intersubunit contacts indicative of the ATP hydrolysis-coupled
motions driving protein unfolding and translocation.
References
[1] Rotanova TV. Bioorg Khim 25, 883 (1999).
[2] Wang N, Maurizi MR, Emmert-Buck L and Gottesman MM J Biol Chem 269, 29308 (1994).
[3] Wang N, Gottesman S, Willingham MC, Gottesman MM and Maurizi, MR Proc Natl Acad Sci USA 90,
11247 (1993).
[4] Lee HS, Kang SG, Bae SS, Lim JK, Cho Y, Kim YJ, Jeon JH, Cha SS, Kwon KK, Kim HT, Park CJ, Lee
HW, Kim SI, Chun J, Colwell RR, Kim SJ and Lee JH J. Bacteriol 190, 7491 (2008).
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S10-5
Novel DNA Ligases with Broad Nucleotide Cofactor Specificity from
the Hyperthermophilic Crenarchaeota
Suk-Tae Kwon
Department of Genetic Engineering, Sungkyunkwan University
DNA ligases fall into two groups on the basis of the required cofactor for activity; ATP-dependent DNA
ligase and NAD+-dependent DNA ligase. The ATP-dependent DNA ligase are widely distributed in eukaryotes
and they are also found in eukaryotic DNA viruses, bacteriophages of the T series and archaebacteria. In
contrast, the NAD+-dependent DNA ligase are found exclusively in eubacteria.
In the past few years, information concerning amino acid sequences and biochemical properties of archaeal
DNA ligase has accumulated. In contrast to the conventional idea that archaea possess only ATP-dependent
DNA ligases, reports of dual specificity DNA ligase from some archaea raised evolutionary issues of cofactor
determinations of DNA ligases.
We assumed that some crenarchaeotal DNA ligases may utilize ADP instead of ATP, while certain
euryarchaeotal DNA ligases may exploit NAD+, those DNA ligases especially belong to the order
Thermococcales. After that, we have reported that DNA ligases from Thermococcus onnurineus NA1 and
Staphylothermus marinus also demonstrated dual cofactor specificity (ATP/NAD+ and ATP/ADP, respectively).
These facts once again prompted us to investigate another crenarchaeotal DNA ligase, Sulfophobococcus zilligii
and Hyperthermus butylicus to analyze and to detect the evolutionary correlation of cofactor requirements of
archaeal DNA ligases.
Unlike other ATP-dependent DNA ligases, and even more distinct from previously reported dual specificity
DNA ligases, the Szi DNA ligase and Hbu DNA ligase exploited ADP, and GTP in comparison to ATP. As we
have confirmed broad cofactor specificity (ATP/ADP/GTP) of the Szi DNA ligase and Hbu DNA ligase, a study
focusing on the evolutionary relationships of DNA ligases on cofactor perspectives was carried out. Similar to
categorization of organisms using 16S rRNA, DNA ligase amino acid sequence alignment also classified
organisms into four distinctive groups; crenarchaeota, euryarchaeota, eukarya, and bacteria. Then, we layered
biochemical properties concerned with respective cofactor requirements of DNA ligases over the tree topology.
Consequently, interesting relationships between cofactor requirements and organism classification was
observed. Eukaryal DNA ligases were more phylogenetically proximate to crenarchaeotal DNA ligase than
euryarchaeotal DNA ligase, while bacterial DNA ligases were relatively closer to euryarchaeotal DNA ligases
than crenarchaeotal DNA ligases in the phylogenetic tree.
In conclusion, it is likely that multiple cofactor specificity crenarchaeotal DNA ligases existed ahead of other
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DNA ligases. Initially, those ancestral DNA ligases utilized ADP, a low free energy source, as a nucleotide
cofactor. So as to seek higher energy sources such as ATP or NAD+, protein structures that interact with those
sources were acquired during the evolutionary process and diverged to eukaryal DNA ligases and
euryarchaeotal DNA ligase. After the emergence of dual specificity ATP/NAD+ DNA ligases in the
euryarchaeota, bacterial DNA ligases obtained NAD+-utilizing ability and subsequently evolved to
contemporary NAD+-dependent bacterial DNA ligase. Broad cofactor specificity of archaeal DNA ligases
seems to have vanished through the evolution and development of organisms in relatively moderate
environments, and are now affixed to either ATP or NAD+-dependent DNA ligase.
So far, Szi DNA ligase and Hbu DNA ligase seem to be the earliest archetype of DNA ligases, since it has
most variant cofactor specificity than any other DNA ligases. Moreover, Szi DNA ligase and Hbu DNA ligase
may exemplify the remnant of an evolution, or the transition phase of cofactor exploitation by showing its
preference to utilize ATP rather than ADP, GTP. This unique property proves its value as evidence of the
evolution of DNA ligase from the perspective of cofactor specificity.
References
[1] Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee J-H, and Kwon S-T. J. Biotechnol. 128, 519 (2007).
[2] Sun Y, Seo MS, Kim JH, Kim YJ, Kim GA, Lee JI, Lee J-H, and Kwon S-T. Environ. Microbiol. 10, 3212
(2008).
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Symposia
S11-1
In Situ Fluorescent Imaging of Bacteriogenic Cyanide in
Lungs of Live Mice Infected with
Pseudomonas aerugionosa and Burkholderia cepacia
Sungsu Park1*, Seong-Won Nam1, Xiaoqiang Chen1, You-Hee Cho2, and Juyoung Yoon1
1
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750
2
Department of Life Science, Sogang University, Seoul 121-742
Cystic fibrosis (CF) patients often suffer from chronic respiratory infections caused by the opportunistic
pathogens Pseudomonas aeruginosa (PA) and Burkholderia cepacia complex (Bcc), which often produce
cyanide (CN- or HCN). Cyanide is a toxin that inhibits cellular respiration. The CF lung exhibits a microaerobic
condition where the cyanogenic pathogens survive through anaerobic respiration and enhance their cyanide
production. A previous report [1] showed that up to 130 µM cyanide was detected from the sputa of CF and
non-CF bronchiectasis patients infected with PA, whereas cyanide was not detected in the sputum of patients
without the PA infection. The presence of cyanide in the sputa from the patients infected with PA can be
interpreted as an indication that cyanide is present in the infected lungs as well. However, the extent of the
cyanide production and the pathogenic role of cyanide in the lungs are mostly unknown because of the lack of a
proper determination method for the in situ concentration of cyanide in the lungs. In vivo imaging offers
noninvasive insight into living organisms and provides spatial and temporal information of the disease-related
changes in the body. Recently, we synthesized a chemosensor which enhances its fluorescence intensity upon
binding CN- and were able to fluorescently visualize the bioaccumulation of cyanide in the nematode
Caenorhabditis elegans using the chemosensor [2].
In this study, the amounts of bacteriogenic cyanide in the lungs of mice infected with the cyanogenic PA or B.
cepacia strains were visualized using the cyanide chemosensor with a whole animal imaging system. For the
chronic infection of PA or B. cepacia in the mouse lungs, BALB/c nude mice were infected through the
intratracheal introduction of agar beads containing these bacteria. For the in vivo imaging, the infected mice
were anesthetized and the cyanide sensor was then injected into their lungs. Using this in vivo imaging method,
we were able to detect high concentrations (1.8 to 4 mM) of cyanide in the lungs of the infected mice, suggesting
that the cyanide production was enhanced in the lungs. Since the in vivo imaging method did not require the
experimental animals to be sacrificed, the long term monitoring of cyanide in the mouse lung was enabled. The
PA and B. cepacia strains continuously produced cyanide in the lungs for at least 9 days and the cyanide
concentration in the lungs was clearly related to the respective bacterial loads during these days. The in vivo
imaging method was used to test the in vivo efficacy of the antibiotics against PA or B. cepacia. PA14 was
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susceptible to the β-lactam (e.g., ceftazidime) and fluoroquinolone (ciprofloxacin) antibiotics, while B. cepacia
was resistant to as these antibiotics. These results suggest that the biogenic cyanide should be considered as an
important virulence factor in the CF lung. In the future, the CFTR (Cystic fibrosis transmembrane conductance
regulator)-deficient mice model should be used to elucidate the pathological role of the bacteriogenic cyanide
against CF patients.
References
[1] B. Ryall, J.C. Davies, R. Wilson, A. Shoemark and H.D. Williams, Pseudomonas aeruginosa, cyanide
accumulation and lung function in CF and non-CF bronchiectasis patients. Eur. Respir. J. 32, 740 (2008).
[2] S.-Y. Chung, S.-W. Nam, J. Lim, S. Park and J. Yoon. A highly selective cyanide sensing in water via
fluorescence change and its application to in vivo imaging. Chem. Commun. 2866 (2009).
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S11-2
Iron Homeostasis Affects Antibiotic-Mediated Bacterial Cell Death
Woojun Park* and Jinki Yeom
Division of Environmental Science and Ecological Engineering, Korea University
Pseudomonas species perform key roles in the environment, including the degradation of natural and
man-made chemicals and the establishment of important interactions with plants and animals [1, 2]. One
pseudomonad, Pseudomonas aeruginosa, is a focus of particular study because it is the predominant
opportunistic pathogen of cystic fibrosis patients [1, 2]. Clearance is difficult because of its unusually low
susceptibility to antibiotic treatment [2, 3]. Thus both because many pseudomonads inhabit natural
environments in which antibiotic exposure is possible, and because P. aeruginosa in particular comprises a
public health hazard, Pseudomonas species are model strains for studies of the development of antibiotic
resistance.
Until recently bactericidal antibiotics were believed to kill cells by several well-established mechanisms,
typically involving the disruption of cell wall biosynthesis, the interruption of DNA replication, or the
overwhelming inhibition of protein synthesis [4, 5]. However, a systems-analysis investigation conducted upon
E. coli by Kohanski, Collins, and co-workers raised the possibility that in this organism bacteriocidal antibiotic
might additionally create oxidative stress, and that a part of their toxicity in aerobic habitats might be due to this
effect [4-6]. We previously discovered that ferredoxin-NADP+ reductase (FprB) is a ferric reductase in P.
putida [7, 8]. Thus we hypothesized that if antibiotic exposure creates H2O2 stress, FprB might promote Fenton
chemistry and contribute to cell death. An fprB mutant will be more resistant to antibiotics than was its
wild-type parent. Conversely, a strain engineered to overproduce FprB will be more sensitive.
In this study, northern blot analysis demonstrated that ahpC, gor, and recA were abundantly induced when
cells were treated with antibiotics (Fig. 1). We also demonstrated directly that iron availability is important to
the action of antibiotics and the ferric reductases of P. putida and P. aeruginosa could accelerate antibioticmediated cell death by promoting the Fenton reaction. The modulation of NADH levels and iron chelation
affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic
resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death (Fig. 2). The results of
transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under
antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron
homeostasis is crucial for bacterial cell survival under antibiotics, and should constitute a significant target for
boosting the action of antibiotics.
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Figure 1. Antibiotics coupled with 100 μM ferric-citrate can
Figure 2. The sensitivity tests were conducted under
increase oxidative stress and DNA repair-related
antibiotic condition using the wild-type, the
gene expression.
fprB mutant strain (∆fprB) and the fprB
References
[1] Gómez MI, Prince A. Curr. Opin. Pharmacol. 7(3), 244-251, 2007.
[2] Gooderham WJ, Hancock RE. FEMS Microbiol. Rev. 33(2), 279-294, 2009.
[3] Page MG, Heim J. Curr. Opin. Pharmacol. 9(5), 558-565, 2009.
[4] Dwyer DJ, Kohanski MA, Collins JJ. Curr. Opin. Microbiol. 12(5), 482-489, 2009.
[5] Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. Cell 130(5), 797-810, 2007.
[6] Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ. Cell 135(4), 679-690, 2008.
[7] Yeom J, Jeon CO, Madsen EL, Park W. J. Bacteriol. 191(5), 1472-1479, 2009.
[8] Yeom J, Jeon CO, Madsen EL, Park W. J. Biochem. 145(4), 481-491, 2009.
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S11-3
Pseudomonas Virulence by a Quorum-Dependently Secreted
Exoprotease
Joon-Hee Lee
Lab of Microbiology, Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 609-735
Pathogenic bacteria use various virulence factors to achieve the invasive infection and hosts defend
themselves against the pathogens by using effective immune systems. Invertebrates mainly depend on innate
immunity that includes the expression of antibacterial peptides and the activation of prophenoloxidase cascades
in hemolymph (insect blood). Generally, these systems are initiated from the recognition of bacteria-specific
materials, so called as PAMPs (pathogen-associated molecular pattern) such as peptidoglycan, lipopolysaccharide,
lipoteichoic acid, and glucan by the cognate specific recognition proteins (pattern recognition receptors). In
insects, signals from the PAMP-recognition are amplified by a proteolytic cascade similar to the vertebrate
complement system, which activates the Toll-mediated intracellular signaling pathway to produce antibacterial
peptides, and the prophenoloxidase system that leads to melanization on the infected site to block further
infection process. While these major innate defense mechanisms of invertebrates are specifically cascaded by
host protease system, bacteria also produce many proteases during infection process, which are known as
virulence factors. A multi-host pathogen, Pseudomonas aeruginosa produces many extracellular proteases
involved in pathogenecity, including elastase (pseudolysin), alkaline protease (aeruginolysin), LasA
(staphylolysin), LasD (staphylolysin), and Protease IV (lysyl endopeptidase). In this study, we investigated the
possibility that bacterial proteases could lead the activation or interrupt of the insect innate immune system by
cleaving the host proteases. When the cell-free culture supernatant (CFCS) of P. aeruginosa was injected into
Tenebrio moliter, the melanization was induced. However, the small molecules passed through the 10 kDa
cut-off membrane or the heat-inactivated CFCS failed to induce the melanization. Interestingly, CFCS from
quorum mutant (lasI-, rhlI-) also failed to induce the melanization and it was restored by the supplement of
synthetic quorum signals during the culture. This suggested that certain quorum-dependently secreted protein
factor(s) may snatch the innate immune response, skipping the initial recognition step. When we mixed the
CFCS with the purified protease cascade components of the T. moliter innate immune system in vitro, some of
them were significantly cleaved into their active forms. In this study, we explored the bacterial proteases
involved in this process and addressed the role of quorum sensing in the Pseudomonas infection and virulence.
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S11-4
Apoptotic Elimination of Natural Killer Cells by
Pseudomonas aeruginosa
Jin Woong Chung
Department of Biological Science, Dong-A University
Infectious complications are one of the major causes of morbidity and mortality in immunocompromised
patients, despite recent advances in therapeutic approaches and supportive care. Among the infectious agents,
the increasing incidence of Pseudomonas aeruginosa (PA) is a worldwide problem, particularly in patients with
leukemia and in hematopoietic stem cell transplantation recipients. PA is a multi-drug resistant, Gram-negative,
opportunistic pathogen and is associated with significant morbidity and mortality. PA constitutes the major
cause of prolonged hospitalization, severe illness, death, and increased cost for immunocompromised patients.
A high mortality rate occurs in patients with underlying disease, such as cystic fibrosis and cancer.
PA pathogenesis involves the production of a variety of toxic products, including alkaline protease (AP),
elastase, and several Type III system-dependent exotoxins that include Exo A, Exo T, and Exo U. AP and
elastase have previously been implicated in the inhibition of NK cell activity, and the exotoxins have been
reported to induce apoptosis of phagocytes, such as dendritic cells, macrophages, and neutrophils. Apoptosis
and shedding of the infected, apoptotic cells may be beneficial to the survival of the host organism. However,
apoptosis of lymphocytes by bacterial infection has detrimental effects on host survival.
NK cells are lymphocytes that mature from hematopoietic stem cells (HSC) in the bone marrow (BM). Upon
activation, they can eliminate leukemic cells, as well as pathogen-infected or transformed cells, either directly
or indirectly through the release of cytokines and chemokines.
Previous studies indicate that upon infection with PA, NK cells can produce interferon-γ that may assist in
clearing the bacteria. However, a negative role of NK cells in the regulation of PA infection has also been
reported. Furthermore, NKG2D and substance P have been shown to be important in host defense against PA
infection, supporting the involvement of NK cells in resistance to such infections. However, little is known
about the exact mechanisms or interactions between NK cells and PA during infection.
In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death
(PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction
in NK cell number. Adoptive transfer of NK cells mitigated PA-induced
metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In
addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to
caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of
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mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data
suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in
immunocompromised patients, such as those with cancer, and provides important insights into the interactions
between PA and NK cells.
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S12-1
Structural Basis for Hypoxia Sensing by Histidine Kinases from
Mycobacterium tuberculosis
Beom Sik Kang
School of Life Sciences and Biotechnology, Kyungpook National University
Mycobacterium tuberculosis is still one of the most dreaded pathogens in existence, and one of the reasons for
its success as a pathogen lies in its ability to persist for years within its host. One-third of the world’s population
is estimated to carry M. tuberculosis in the dormant form [1], and while in this state, the pathogen is insensitive
to most available chemotherapy. M. tuberculosis has been shown to undergo a metabolic transformation to its
non-replicating persistence state under the influence of environmental stimuli such as hypoxia or nitric oxide [2].
This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT [3, 4], each of
which consists of a sensor core and a kinase core [5]. The sensor core contains two GAF domains that are
responsible for detecting oxygen tension.
The first GAF domain (GAF-A) contains a heme [6] while the second GAF domain (GAF-B) is not suitable
to bind a small ligand such as cyclic nucleotides [7]. In the crystal structure of the first GAF domain (GAF-A) of
DosS, a b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly
perpendicular to the β-sheet of the GAF domain. The heme iron was liganded by H149 at the proximal heme
axial position. The iron, in the oxidized form, was six-coordinated with a water molecule at the distal position.
Upon reduction, the iron, in ferrous form, was five-coordinated and when the GAF domain was exposed to
atmospheric O2, the ferrous form was oxidized to generate the met form rather than a ferrous O2-bound form.
Since the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen
bond network found at the heme site could accelerate the electron transferability and would explain why DosS
was unable to bind O2. Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was
unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its
reduction.
The kinase core of histidine kinases contains an ATP-binding domain following a histidine kinase
phosphor-acceptor (HisKA) domain. The ATP-binding domains have been known to contain conserved motifs,
N, F, and three G boxes [8]. However, sequence alignment analysis revealed that DosS and DosT do not have F
and G2 boxes, which play a role for ATP lid. Crystal structures of ATP-binding domains from DosS and DosT
show a closed ATP-binding pocket with a short putative ATP-lid. The crystal structures suggest that the
ATP-binding domain should undergo a conformation change, which opens the ATP binding site, probably by
the sensor domain, GAF-A.
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References
[1] Parrish NM, Dick JD, and Bishai WR. Trends Microbiol 6, 107 (1998).
[2] Wayne LG and Sohasjey CD. Annu Rev Microbiol 55, 139 (2001).
[3] Dasgupta N, Kapur V, Singh KK, Das TK, Sachdeva S, Jyothisri K, and Tyagi JS. Tuber Lung Dis 80, 141
(2000).
[4] Roberts DM, Liao RP, Wisedchaisri G, Hol WGJ, and Sherman DR. J Biol Chem 279, 23082 (2004).
[5] Sardiwal S, Kendall SL, Movahedzadeh F, Rison SCG, Stoker NG, and Djordjevic S. J Mol Biol 353, 929
(2005).
[6] Cho HY, Cho HJ, Kim YM, Oh JI, and Kang BS. J Biol Chem 284, 13057 (2009).
[7] Lee J, Cho HY, Cho HJ, Ko I, Park SW, Baik H, Oh J, Eom C, Kim YM, Kang BS, and Oh J. J Bacteriol
190, 6795 (2008).
[8] Nowak E, Panjikar Santosh, Morth JP, Jordanova R, Svergun DI, and Tucker PA. Structure, 14, 275
(2006).
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S12-2
Structural Studies of the Transmembrane Domain of
Bacterial Histidine Kinase
Eunha Hwang1, Young-Pil Kim1, Kwonjoo Yeo1, Innokentiy V. Maslennikov2, Christian Klammt2,
Georgia Kefala2, Chaejoon Cheong1, Senyon Choe2, and Young Ho Jeon1*
1
Division of Magnetic Resonance, Korea Basic Science Institute, Ochang 363-883
2
Structural Biology Lab., Salk Institute, USA
Histidine kinase plays an important role in signal response to the external stimuli in bacteria. Currently, the
structural studies of the transmembrane domain of histidine kinase are deficient because of the difficulties of the
sample preparation. The main difficulties include the low expression of the membrane protein and the
inhomogeniety of the sample state. Here, we show the successful expression of transmembrane domain (PTD)
of bacterial histidine kinases, with fusion of Vitreoscilla hemoglobin (VHb), and the sample preparation of
selected proteins in mg quantities for nuclear magnetic resonance (NMR) studies. The PTDs of the histidine
kinases were then screened with various conditions to obtain homogeneous NMR spectra with well-resolved
resonances. We achieved the near-complete backbone assignments (>90%) of the two intergral membrane
proteins of DPC micelles and LMPG micelles. These results may provide a valuable opportunity and
information for the structural study by NMR.
Fig. 1. Possible topology models of membrane proteins in detergent micelles.
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References
[1] Kim YP, Yeo KJ, Kim MH, Kim YC*, Jeon YH* (2010) Structural characterization of the intra-membrane
histidine kinase YbdK from Bacillus subtilis in DPC micelles, Biochem Biophys Res Commun.
391(3):1506-11.
[2] Kim HJ, Howell SC, Van Horn WD, Jeon YH, Sanders CR (2009) Recent Advances in the Application of
Solution NMR Spectroscopy to Multi-Span Integral Membrane Proteins, Prog Nucl Magn Reson
Spectrosc. 55(4):335-360.
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S12-3
Functional Characterization of Native and Recombinant Antifreeze
Protein of Arctic Yeast, KOPRI-AY30
Kyung Sun Park1,2, Jong Kyu Lee1, Jong Soo Kim1, and Hak Jun Kim1,2*
1
Division of Polar Biology and Ocean Sciences, Korea Polar Research Institute, Incheon 406-840
2
Department of Polar Sciences, University of Science and Technology, Incehon 406-840
Antifreeze proteins (AFPs) are proteins that depress the freezing point but not the melting point of aqueous
solutions by inhibiting the growth of ice crystals. This phenomenon is described as thermal hysteresis (TH), a
difference between the freezing and melting points and provides organisms in subzero environments with one of
cold adaption mechanisms. These characteristics make AFPs a potential biomedical, food, agricultural, and
industrial applications.
Recently, we have isolated AFPs from an Arctic psychrophilic yeast Leucosporidium sp. using ice-affinity
chromatography, which showed antifreeze activity as well as ice recrystallization inhibition activity. To further
characterize the protein, recombinant AFP was expressed and purified. However, the native AFP was observed
to be N-glycosylated. To see the activity of the glycosylated AFP in relation to their structure, we conducted
several experiments, such as, dynamic light scattering, circular dichroism, chemical cross-linking, and heat
stability.
The secondary structural analysis by CD spectra of native AFP showed minima around 200 nm, a typical
characteristic of random coil protein while, recombinant AFP showed increased a-helicity. In Glutaraldehyde
cross –linking experiment, recombinant AFP was prone to form oligomers esp. dimer but native AFP was not.
Heat stability of the both proteins showed little difference although recombinant AFP seemed more stable.
Effect of glycosylation on behavior and function of AFPs seemed to be negligible but yeast AFP seemed not to
be case. Here, we will present the effect of glycosylation of yeast AFP in terms of antifreeze activity.
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S12-4
From Oxylipin to Volatile
Dongsun Lee
Jeju National University
The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs),
which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and
hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and
GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different
products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with
substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity
of an epoxyallylic radical and its cation by means of interactions with an aromatic p-system. Replacing the
amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is
both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining
our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown
hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in
amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of
plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and
Cephalochordata.
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S12-5
Spectroscopic Study of Rhodopsins in Action
Hideki Kandori
Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan
We have studied structure-function relationship in visual and microbial rhodopsins by means of various
spectroscopic techniques. In particular, light-induced difference Fourier-transform infrared (FTIR)
spectroscopy is a promising method for this aim [1]. In the symposium, I will present on the microbial
rhodopsins that function as light-driven ion pumps.
(1) Mechanism of light-driven ion pumps
Proton transport is essential for our life, because ATP is synthesized by an enzyme that utilizes proton motive
force, and thus nature creates various proton pumps. Proton pump proteins can translocate protons even when
the proton concentration is higher at the other side of the membrane. Proton pathways are necessary for pumps,
but proton pathways cannot be fully connected between the two sides of the membrane, because the proton
gradient formed will be easily collapsed. This is an important aspect in distinguishing pumps from channels.
The former needs a "switch", which ensures the vectoriality of the pumping. As the heart of pumps, the
switching machinery is interesting, but little is known about it.
Halophilic bacteria possess two light-driven pumps; bacteriorhodopsin (BR) and halorhodopsin (HR) pump
protons and chloride ions, respectively. BR is the best understood
ion pump, whose structure and structural changes have been
extensively investigated. Recently, we succeeded in visualizing the
real-time motion of BR using high-speed Atomic Force
Microscope (AFM) [2]. Outward proton transport is achieved by 5
proton transfers, and each proton transfer is spatially and
temporally well controlled (Figure). By means of low-temperature
FTIR spectroscopy, we revealed the presence of internal water
molecules of BR and their important role in function before the
X-ray structural determination [3]. In the active center of BR, we
found strongly hydrogen-bonded water molecules (water O-D
stretch in D2O at <2400 cm-1), and comprehensive study of various
rhodopsins (>15 original papers including the latest one on
Gloeobacter rhodopsin [4]) revealed that such water molecules
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were found only in the proteins exhibiting proton-pumping activity [5]. We conclude that a single hydrogen
bond of water is possibly the determinant of the proton-pump function in rhodopsins.
(2) Functional conversion of light-driven ion pumps
Functional conversion is important for both basic and application studies. We previously converted BR into
an inward chloride-ion pump [6], but interestingly, HR has never been converted into proton pump. This also
suggests the importance of hydrogen-bonding network in the active center.
An inward proton pump has never been created naturally or artificially. This sounds reasonable, because
inward proton pump competes the ATP synthase function, being dangerous for survival. Proton pumps must
have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the
case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Recently, we succeeded
in creating an inward-directed proton transport from a bacterial rhodopsin by a single amino acid replacement
[7]. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little
proton pump activity. When we replace Asp217 at the cytoplasmic domain (distance ~15 Å from the retinal
chromophore) to Glu, ASR exhibited an inward proton transport activity. FTIR spectra clearly show an
increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal
proton pumps. The newly designed inward proton transport may be useful as an application tool in neurobiology
as well as channelrhodopsin, a light-activated cation channel, and HR.
References
[1] H. Kandori, Retinal binding proteins. in cis-trans Isomerization in Biochemistry; Wiley-VCH: Freiburg,
pp 53-75 (2006).
[2] M. Shibata, H. Yamashita, T. Uchihashi, H. Kandori and T. Ando, Nature Nanotechnology 5, 208 (2010).
[3] H. Kandori, Biochim. Biophys. Acta 1460, 177 (2000).
[4] K. Hashimoto, A. R. Choi, Y. Furutani, K.-H. Jung and H. Kandori, Biochemistry in press (2010).
[5] Y. Furutani, M. Shibata and H. Kandori, Photochem. Photobiol. Sci. 4, 661 (2005).
[6] J. Sasaki, L. S. Brown, Y.-S. Chon, H. Kandori, A. Maeda, R. Needleman and J. K. Lanyi, Science 269, 73
(1995).
[7] A. Kawanabe, Y. Furutani, K.-H. Jung and H. Kandori, J. Am. Chem. Soc. 131, 16444 (2009).
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S13-1
Marine Brown Kelp Diversity Based on the Analyses of Multigenes
from Plastid and Mitochondrion
Ga Youn Cho
Division of Non-Vascular Plants, National Institute of Biological Resources, Incheon 404-708
Brown algal kelp species are the largest seaweeds and provide a variety of microhabitats for marine
organisms and are often used for food and medicine. A robust molecular systematic framework is yet to be
established for these species largely due to insufficient taxon sampling. The present study aimed to examine
molecular diversity, phylogenetic relationship, and evolution of the kep based on nucleotide sequence
information. Here, we analyzed 42 species from 27 genera, which represent all genus-level taxa in the
Laminariales described to date except Undariella and Feditia. Five protein-coding plastid genes (i.e., psaA,
psbA, psbC, tufA, and rbcL) and a mitochondrial gene (i.e., cox3) were analyzed using phylogenetic methods.
All trees from individual and combined data sets were congruent and supported the monophyly of all kelps.
Within the Laminariales, 11 well supported lineages were identified by our study. These results do not support
the long-held morphological classification of Laminariales into three families and 10 tribes. Instead, we propose
a taxonomic revision of the Laminariales which is defined by a single family (i.e., the Laminariaceae) and 11
tribes.
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S13-2
Functional Analysis in Escherichia coli of Biosynthesis Genes for
Isoprenoids, Carotenoids, and Sesquiterpenes, and Their Production
with Higher Plants
Norihiko Misawa
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa
921-8836, Japan ([email protected])
Structural and functional diversity of isoprenoids
Isoprenoids, also referred to as terpenes, are the most diverse class of natural products consisting of more than
23,000 structurally different compounds, which have been isolated from a variety of natural sources including
higher plants and microorganisms. Since these compounds have a wide range of biological functions, they have
extensively been applied to pharmaceuticals, nutraceuticals, fragrances, flavorings, cosmetics, colorants, or
agrichemicals. For example, a monoterpene (C10) linalool, which is included in the essential oil of hop
(Humulus lupulus), is an inevitable aroma ingredient for beer brewery. A sesquiterpene (C15) artemisinin, which
is extracted from the shoots of annual wormwood Artemisia annua, has been used as an antimalarial drug. Taxol
(paclitaxel), a diterpene (C20) including three benzene rings in the molecule, isolated from Pacific yew (Taxus
brevifolia), has been used as an effective anti-cancer drug. Glycyrrhizin is a triterpene (C30) glycoside
(triterpene saponin), extracted from licorice (the root and stolon of the Glycyrrhiza plants), is widely used as a
natural sweetener as well as a pharmaceutical agent. Ginsenosides, a series of triterpene glycosides that are
extracted from ginseng (the root of Panax ginseng), have been a traditional medicine due to their various
beneficial effects on human health. Carotenoids (tetraterpenes; C40), which include astaxanthin,
β-cryptoxanthin, lutein, zeaxanthin, lycopene, and β-carotene, have attracted great attention due to their
beneficial effects on human health, e.g., their prevention of chronic diseases such as cancer, cardiovascular
disease, and age-related macular degeneration, and used commercially as nutraceuticals for pharmaceutical and
cosmetic purposes.
Biosynthesis of isoprenoid basic structures
Biosynthetic routes to isoprenoids are regularly and “simply” organized, despites their enormous structural
diversity. They are all biosynthesized from the same basic isoprene units, isopentenyl diphosphate
(pyrophosphate) (IPP) and its isomer dimethylallyl diphosphate (DMAPP) (Fig. 1). Most prokaryotes including
Escherichia coli and plant plastids synthesize IPP and DMAPP through the non-mevalonate [2-C-methyl-Derythritol 4-phosphate (MEP)] pathway starting with the reaction between pyruvate and glyceraldehyde
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3-phophate. DMAPP and IPP are condensed to generate geranyl diphosphate (GPP; C10), which is further
converted with IPP into farnesyl diphosphate (FPP; C15), with FPP synthase (Fig. 1). FPP is similarly condensed
with IPP to form geranylgeranyl diphosphate (GGPP; C20) with GGPP synthase that is CrtE in the case of
carotenogenic (carotenoids-producing) bacteria. GPP and FPP are the precursors to volatile mono- and
sesquiterpenes, respectively. GGPP is the precursor for diterpenes including gibberellins. Two molecules of
FPP and GGPP are typically condensed to squalene and phytoene, which are the first substrates of triterpenes
and carotenoids, respectively.
Monoterpenes
OPP
IPP
FPP synthase
OPP
IPP isomerase
(type 1, 2)
DMAPP
IPP
OPP
GPP
PPi
IPP
GGPP synthase
PPi
OPP
OPP
GGPP
(2 X GGPP)
FPP synthase
PPi
Diterpenes
Carotenoids
(Tetraterpenes)
IPP
FPP
(2 X FPP)
Triterpenes
Sesquiterpenes
Fig. 1. Biosynthesis of isoprenoid basic structures.
Functional analysis of isoprenoid biosynthesis genes
E. coli has been found to be the microorganism of choice for many cases of the functional analysis of a
biosynthesis genes as well as biotechnological applications. This is because extensive molecular genetic
resources are present and it is amenable to genetic manipulation, and considerably tolerant to solvents. Since
1990, the functional analyses of carotenoid biosynthesis genes have successfully been performed using E.
coli.1,2 Recently, the functional analysis system for sesquiterpene synthase (cyclase) genes were developed
using recombinant E. coli cells, in which the Streptomyces mevalonate pathway genes were co-expressed for the
utilization of D-mevalonate (D-mevalonolactone) supplemented in the culture medium.2 Various sesquiterpene
synthase genes from higher plants, which include the genes for α-humulene synthase, β-eudesmol synthase, and
(S)-β-bisabolene synthase from the genus Zingiber, have been identified using this system.
Biotechnological production of carotenoids with higher plants
Many commercially relevant isoprenoids are present in minute quantities in nature or low yielding from their
natural sources. Chemical synthesis of these isoprenoids has been an important target for chemists, although it is
often difficult or costly because of their structural complexity. Biosynthesis of such isoprenoids with genetically
modified heterologous hosts should be an alternative and promising approach. Biotechnology researches
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towards efficient production of the isoprenoids have extensively been carried out using recombinant bacteria
such as Escherichia coli and yeasts such as Saccharomyces cerevisiae, and using transgenic plants.
We have performed genetic engineering of oil crops, rape (canola; Brassica napus), one of the major oil crops
worldwide that includes large amounts of oleic acid (18:1), and linseed flax (Linum usitatissimum), an
industrially important oil crop that includes plenty amounts of α-linolenic acid (18:3), to increase their
carotenoid contents and to produce such industrially useful carotenoids in the seeds. The phytoene synthase
(crtB) gene derived from soil bacterium Pantoea ananatis (formerly classified as Erwinia uredovora) was
introduced into flax seeds under the control of the fatty acid elongation enzyme 1 gene (FAE1) or CaMV 35S
promoter. Subsequent transgenic flax seeds synthesized 0.16 mg/g fresh weight of carotenoids as the total
amounts (at the maximum), which corresponded to 19-fold increase compared with that of an untransformed
control. The carotenoid composition was β-carotene, α-carotene, phytoene, and lutein. We expressed multiple
(max seven) key genes for ketocarotenoid biosynthesis in rapeseed, which were the crtW and crtZ genes
originated from marine bacterium Brevundimonas sp. strain SD212, idi from marine bacterium Paracoccus sp.
N81106, and the P. ananatis crtE, crtB, and crtI genes. The transgenic rape plants were enriched with large
amounts of carotenoids (1.37 mg/g fresh weight; corresponded to 39-fold increase at the maximum) in the seeds,
which included ketocarotenoids such as astaxanthin, canthaxanthin and echinenone in addition to β-carotene,
α-carotene, phytoene, β-cryptoxanthin, and lutein.
References
[1] Y. Nishida et al., Appl. Environ. Microbiol., 71: 4286-4296, 2005.
[2] H. Harada and N. Misawa, Appl. Microbiol. Biotechnol., 84: 1021-1031, 2009.
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S13-3
5-Hydroxyindole-Type Alkaloids from Marine Organisms
Hyi-Seung Lee*, Hee Jae Shin, and Yeon Ju Lee
Marine Natural Products Laboratory, Korea Ocean Research and Development Institute, Ansan 425-600
Much attention has been focused on indole alkaloids owing to their potential utility and significant biological
activity, including cytotoxic, antiviral, antimicrobial, antiparasitics, and anti-inflammatory. A variety of marine
sources including sponges, tunicates, red alga, and symbiotic bacteria have been shown to generate indole
alkaloids, which represent the largest number and most complicated of the marine alkaloids [1]. The alkaloids
obtained from marine organisms frequently possess novel frameworks while in other cases terrestrially related
compounds clearly exist. Their structure elucidation, chemical modification, and synthesis have received a great
deal of interdisciplinary attention from areas of research other than chemistry and include pharmacology and
medicine.
In the course of our study on the chemical investigation of marine organisms [2], we found indol alkaloids
from the Micronesian sponge Hyrtios sp. collected in Chuuk State, Federated States of Micronesia and Korean
sponge Psammocinia sp. collected in Dokdo Island, Korea. Bioassay-guided separation of the crude extracts using
various chromatographic techniques yielded a new β-carboline alkaloid together with known 5-hydroxyindol
alkaloids. From the extracts of Hyrtios sp., 1-carboxy-6-hydroxy-3,4-dihydro-b-carboline (1), 6-hydroxy-3,4dihydro-1-oxo-β-carboline (2), 5-hydroxy-1H-indole-3-carboxylic acid methyl ester (3), serotonin (4), hyrtiosin
A (5), 5-hydroxyindole-3-carbaldehyde (6), and hyrtiosin B (7) were isolated. And from the extracts of
Psammocinia sp., bis-indole alkaloid, hyrtinadine A (8) together with the known metabolites were isolated.
Their structures were elucidated on the basis of mass spectrometry and detailed 2D NMR spectroscopic data.
The isolated compounds were evaluated for their inhibitory activities against ICL of C. albicans. The inhibitory
potencies, expressed as IC50 values, of the tested compounds are compared to that of a known ICL inhibitor,
3-nitropropinate. Among the compounds tested, hyrtiosin B (7) was found to be a strong ICL inhibitor.
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To investigate the structure-activity relationship on the inhibitory activities, the indol derivatives were
synthesized by the Pitet-Spengler reaction. All of the synthesized compounds were tested for the inhibitory
activities. The preliminary structure-activity relationship, to elucidate the essential structure requirements for
the inhibitory activity, will be described.
References
[1] J. Kobayashi, T. Murayama, M. Ishibashi, S. Kosuge, M. Takamatsu, Y. Ohizumi, H. Kobayashi, T. Ohta,
S. Nozoe, and T. Sasaki. Tetrahedron 46 (1990) 7699.
[2] H.-S. Lee, K.-M. Yoon, Y.-R. Han, K. J. Lee, S.-C. Chung, T.-I. Kim, S-H. Lee, J. Shin, and K.-B. Oh.
Bioorg. Medi. Chem. Lett. 19 (2009) 1051.
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S13-4
Marine Microbial Water Quality off Hawaii
Hans Krock, Ph.D., P.E.
Emeritus Professor, University of Hawaii
Over the last four decades, studies in the deep waters off the Hawaiian Islands have defined several
parameters relevant to understanding the dynamics of the microbial community in these waters. These studies
were related to ocean thermal energy conversion (OTEC) research projects and to the disposal of municipal
wastewater.
The mid-ocean location of Hawaii, away from direct continental influences, allowed definition of the
environmental conditions found in the open ocean – a large area of the world that has not been extensively
studied with respect to microbiology. The OTEC related studies have helped define the essentially pristine deep
ocean and mid-water environment while the studies related to municipal wastewater show the extent of the
influence from wastewater discharges on the ocean surface layer environment near a tropical island.
The deep ocean water, below the thermocline and above the influence of the bottom, was found to be very
clear (turbidity ~ 0.03 NTU) with low levels of dissolved oxygen and essentially no influence from human
activities. The ambient microbial population of these waters has not been studied in any significant detail.
Modest concentrations of microbial colonies can be detected using heterotrophic plate counts. However, under
in situ conditions, most of these bacterial populations are likely to not be metabolically active since there are
almost no reduced organics in these waters. Detailed study and identification of the microbiological populations
of these waters remains to be done. Such an effort will likely accompany the development of the thermal energy
resource of the tropical ocean using OTEC.
Much more is known about the microbial population of the nearshore tropical oceanic surface layer. In the
mid-ocean, these waters are generally nutrient limited but, because of good mixing and transport, do not have a
significant eutrophication response to municipal wastewater discharges located on open coastlines outside of
embayments. Surface waters subject to solar UV radiation are relatively rapidly disinfected with respect to
human pathogens and their indicator organisms. However, long term (days and weeks) wastewater related
microbial contamination can be detected in these waters below the depth of UV penetration. UV disinfection of
the wastewater before discharge effectively controls this contamination.
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S13-5
Plant Growth-Promoting Fungi from the Sand-Dune
Plants in East Coast of Korea
Yung-Hyun You1, Sumera Afzahl Khan6, Jung-Sook Hwang2, Yeon-Sik Choo2,
In-Jung Lee3, Sang-Dal Kim5, In-Koo Lee4 and Jong-Guk Kim1*
1
School of Life Science and Biotechnology, 2Department of Biology, 3Department of Agronomy,
4
Department of Agricultural Chemistry, Kyungpook National University, Taegu 702-010
5
Yeungnam University, Gyeongsan
6
Center of Biotechnology, University of Peshawar, Pakistan
About half of sand dunes in coastal regions of South Korea are being destroyed due to anthropogenic
disturbances such as military action as well as beach construction and recreational activities. The intensity of
artificial activities had affected the speed and efficiency of their conservation and revegetation. We investigated
root endophytic fungi of sand-dune flora for GAs production as such work had not been carried out in the past.
1,438 fungal strains were isolated from the root of sand-dune plant, and 3 strains were selected by plant
gowth-promoting activity. One of these strain was identified as Penicillium citrinum KACC43900. Analysis of
the culture filtrate of P. citrinum showed the presence of physiologically active gibberellins, GA1, GA3, GA4
and GA7 (1.95 ng/ml, 3.83 ng/ml, 6.03 ng/ml and 2.35 ng/ml, respectively) along with other physiologically
inactive GA5, GA9, GA12, GA15, GA19, GA20 and, GA24. The plant growth promotion and gibberellin producing
capacity of P. citrinum was much higher than the wild type Gibberella fujikuroi, which was taken as control
during present study.
When a sand dune plants were treated with the culture filtrate of P. citrinum KACC43900, their growth were
promoted and the rate of photosynthesis was increased. And expression level of gibberellins 3-oxidase,
cytochrome P450 family protein and ent-kaurene synthase was increased.
Therefore P. citrinum KACC43900 can be used for growth promotion of sand-dune plants in destructed sea
sand-dunes.
References
[1] S. A. Khan et al, Biotechnol Lett (2009) 31:283-287.
[2] S. A. Khan et al, BMC Microbiology (2008) 8:231 doi:10.1186/1471-2180-8-231.
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122
Symposia
2010 International Meeting of
the Microbiological Society of Korea
Colloquium
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C-1
Pyrene Metabolism and Enzymes Involved in
Novosphingobium pentaromativorans US6-1
Yuanrong Luo1*, Sung Ho Yun2, Kae Kyoung Kwon1, Sang Jin Kim1,
Seung Il Kim2, and Young Ho Chung2
1
Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, Ansan 426-744
2
Korea Basic Science Institute, Daejeon 305-333
Pyrene is a polycyclic aromatic hydrocarbon (PAH) commonly present in PAH-contaminated sites.
Novosphingobium pentaromativorans US6-1 was isolated from sediment of Ulsan Bay and has broad substrates
with two to five rings PAHs. Newly and increased expression of proteins were found with 1-D PAGE or 2-DE
and LC-MS/MS. Presence of pyrene 1,2-monooxygenase, pyrene 4,5-monooxygenase and 4,5-dihydroxypyrene
dioxygenase indicates that degradation of pyrene in N. pentaromativorans US6-1 proceeds via multiple
metabolic routes initiated by mono-(C-1,2 and C-4,5) and dioxygenation (C-4,5) reactions. Further degradation
entered phenanthrene metabolic pathway and via either o-phthalate pathway or salicylate pathway, both
pathways were subsequently entered tricarboxylic acid (TCA) cycle and mineralized to CO2. A pyrene
degradation pathway for N. pentaromativorans US6-1 was proposed by identifying the enzymes involved in the
pyrene degradation [Supported by MEGRC].
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C-2
Microarray Analysis of Knockout Mutant LuxS Relevant to the
Quorum Sensing Mechanism in Escherichia coli K-12
Su-Wan Son1*, Jaisoo Su Kim1, Yong-Hyun Cho2, Ji-Youl Lee2, Seung-Ju Lee2, Kyong-Ran Peck3,
and Sang-Seob Lee1
1
Department of Biological Engineering, Kyonggi University
Department of Urology, School of Medicine, The Catholic University
3
Division of Infectious Diseases, Sungkyunkwan University School of Medicine
2
The LuxS synthesized an autoinducer to relate the quorum sensing (QS) mechanism. These autoinducer was a
signal molecule and it preformed a biofilm from cell to cell community. In this study, we investigated that it was
effect on the QS and virulence related gene expression level. First, we made the LuxS knockout mutant by
pDM4 vector system. To investigate the role of luxS in Escherichia coli k-12, a luxS mutant strain was
constructed by integration of the suicide plasmid (pDM4 vector) containing the kanamycin resistance gene into
the chromosomal luxS gene. This mutant could be confirming to polymerase chain reaction (PCR) of genomic
DNA and thin layer chromatography (TLC). N-acylhomoserine lactone (AHL) signal molecules were detected
in E. coli K-12 strain with the Agrobacterium tumefaciens reporter strain NT1 (pDCI4IE33). On the basis of this
mutant, we experimented on the Microarray for a study on the effect of luxS gene in the E. coli k-12. It was
found from the results that luxS gene is an important factor in QS and virulence mechanism. Further research on
the microarray results would clarify the correlation of QS and virulence mechanism.
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C-3
Osmotic Stress Inhibits the Cell Growth of Listeria monocytogenes
by the Downregulation of PTS Genes
Dongryeoul Bae* and Chinling Wang
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State,
Mississippi 39762, USA
The concern regarding Listeria monocytogenes is becoming increased due to their adaptable abilities and
inherent resistances to environmental stresses. Osmotic stress can damage the bacterial cells via the disruption
of the bacterial maintenance of osmotic pressure. The molecular mechanisms that L. monocytogenes adapt to
high osmolarity are not fully understood. In order to examine the gene expression profiles of L. monocytogenes
strain F2365 to osmotic stress, cDNA microarray was used. We have characterized genes mediated from the
strain grown in BHI medium supplemented with 1.2% NaCl at the transcriptional level. Four and twenty four
genes were upregulated and downregulated by the stress, respectively. The data from cDNA microarray
analysis were confirmed by quantitative real time RT-PCR. Based on microarray data, the expression level of
genes encoding ribosomal protein L2 and the ATP-binding cassette superfamily proteins involved in the uptake
of glycine betaine/L-proline were upregulated, whereas the expression level of genes associated with PTS
system, its metabolic enzymes, pathogenesis, and hypothetical protein were downregulated. The growth of L.
monocytogenes in the medium with salt was significantly inhibited (P<0.01). The expression level of PTS
transport genes involved in the uptake of glucose, fructose, mannose, and cellobiose were examined at various
concentrations of NaCl. All genes were significantly downregulated, suggesting that osmotic stress may inhibit
listerial cell growth through the downregulation of genes involved in sugar uptake. The current study reports
global gene expression level in F2365 to osmotic stress. In advance, this study provides a baseline for the
mechanism of the growth or osmotolerance of L. monocytogenes.
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C-4
Direct Activation of Transcription of Fur-Positive Regulon by
Iron-Free Form of Fur Protein
Hyun-Jung Lee
Dept. Environ. Sci., Hankuk Univ. Foreign Studies and
Dept. Environ. Med. Biol., Yonsei Univ. Coll. Med.
Pathogenic bacterial ability to acquire iron from host environments is essential to maintain its growth and to
elicit its virulence, which is mainly regulated by Fur. Recently, expression analysis of Vibrio vulnificus fur gene
indicated that Fur acts as a transcription activator under the iron-deprived condition. This finding led us to
question if this novel regulation is unique in autoregulation of fur expression, or it is widely distributed in V.
vulnificus genome. Thus, the DNA microarray was utilized to screen the ORFs activated by Fur under
iron-limited conditions. EMSA showed that the iron-free Fur directly and specifically bound the upstream
regions of each candidate gene. DNase I footprint assays identified its binding sites, which located around -150
relative to the corresponding transcription start sites. Fusion assays showed that in vivo expression of each
fusion in wildtype was increased in the presence of an iron-chelator, and this positive effect was dependent upon
the presence of functional Fur protein. The requirements of the binding sites for activation by iron-free Fur were
further examined via site-directed mutagenesis of the sites. Therefore, this study demonstrates that V. vulnificus
Fur protein is dynamically involved in transcription of diverse genes via repression by iron-complex form and
activation by iron-free form.
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2010 International Meeting of
the Microbiological Society of Korea
Workshop
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130
Workshop
W1-1
Microbial Community Analysis Using Pyrosequencing
(Pyrosequencing을 이용한 미생물 군집 분석)
Jongsik Chun
CEO, ChunLab, Inc.
시료 안에 존재하는 미생물의 종류와 양을 정확히 측정하는 것은 직접적인 연관이 있는 생태학자뿐만
아니라, 미생물학자에게 중요한 기술이다. 그 동안 denaturing gradient gel electrophoresis (DGGE)와 같은
간접적인 군집분석 기술이 많이 쓰여 왔으나, Next generation sequencing (NGS) 기술의 도래로 직접적인
DNA sequencing 에 의한 군집의 분석이 가능해 졌다. NGS 기술 중에서도 Pyrosequencing 은 400 bp 이상의
DNA 염기서열의 해독이 가능하고, 그 throughput 도 커서 Bacteria, Archaea, Fungi 등의 다양한 미생물의 군집
분석이 가능하다. 본 워크숍에서는 다음과 같은 내용으로 Pyrosequencing 을 이용한 미생물 군집 분석에 대해
소개하고자 한다.
(1) Next generation sequencing 기술의 개요
(2) Pyrosequencing 원리
(3) 미생물 군집 분석 전략 (Phylogenetic marker, primer design 등)
(4) 통계 처리의 문제점과 해결 전략
(5) 통합 분석 소프트웨어 소개
(6) Human microbiome, 물환경 시료, 식품 등의 분석 적용 예
(7) Genome 분석, Comparative genomics, Metagenomics 분야에서의 Pyrosequencing 적용 전략
131
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124
Colloquium
2010 International Meeting of
the Microbiological Society of Korea
Poster Sessions
A. Systematics
B. Ecology and Environmental Microbiology
C. Applied Microbiology
D. Immunology and Microbial Pathogenesis
E. Physiology and Biochemistry
F. Genetics
G. Biotechnology
H. Others
133
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134
Poster Sessions
A001
A003
Brachybacterium squillarum sp. nov., Isolated from
Salt-Fermented Seafood
Pedobacter yonginensis sp. nov., Isolated from a
Mesotrophic Artificial Lake in Korea
Seong-Kyu Park*, Min-Soo Kim, Mi-Ja Jung, Eun-Jin Park,
Young-Do Nam, Seong Woon Roh, and Jin-Woo Bae
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University
Yochan Joung*, Haneul Kim, and Kiseong Joh
Department of Bioscience and Biotechnology,
Hankuk University of Foreign Studies
A Gram-positive bacterium, strain M-6-3T, was isolated from
salt-fermented seafood in Korea. The organism grows in
conditions ranging from 0-10% (w/v) NaCl, and 25-37°C, with
optimal growth occurring at 5% NaCl and 28-30°C. The polar
lipid profile of M-6-3T consists of diphosphatidylglycerol
(DPG), phosphatidylglycerol (PG), an unidentified phospholipid
(PL), and an unknown glycolipid (GL). Strain M-6-3T contains
MK-7 as the major component of the quinone system and
anteiso-C15:0 (62.1%) as the predominant fatty acid. Based on 16S
rRNA gene sequence similarity, strain M-6-3T is most closely
related to Brachybacterium rhamnosum LGM 19848T (98.5%).
The G+C content of the genomic DNA was measured at 71.5
mol% and DNA-DNA hybridization values were under 10%
with reference strains. Based on phenotypic, genotypic and
phylogenic analyses, the name Brahcybacterium squillarum sp.
nov. is proposed for strain M-6-3T (= KACC 14221T = JCM
16464T).
A002
A non-motile and red-pigmented bacterium, designated strain
HMD1002T, was isolated from an artificial lake located within
the campus of Hankuk University of Foreign Studies, Korea.
The major fatty acids were iso-C15 : 0 (29.6%), Sum In Feature
3 (comprising C16 : 1 ω7c and/or iso-C15 : 0 2-OH;17.5%), isoC17 : 0 3-OH (12.5%), 15:1 w6c (8.3%), Sum In Feature 9
(comprising iso- C17:1 -w9c:6.9%), C15 : 0 (5.3%), iso-C15 : 0
3-OH (2.6%), anteiso-C15:0 (2.4%). The DNA G+C content
was 41.0 mol%. A phylogenetic tree based on 16S rRNA gene
sequences showed that strain HMD1002T formed a lineage in
the genus Pedobacer and was closely related to Pedobacer
terrae (96.3% sequence similarity) and Pedobacer suwonensis
(95.8% ). On the basis of the evidence presented in this study,
strains of HMD1002T represent a novel species of the genus
Pedobacter, for which the name Pedobacter yonginensis sp. is
proposed. The type strains are HMD1002T (=KCTC 22721T=
CECT 7544T).
A004
Leucobacter celer sp. nov., Isolated from Korean
Fermented Seafood
Gramella jeungdonese sp. nov., Isolated from Saltern
in Korea
Na-Ri Shin*, Min-Soo Kim, Mi-Ja Jung, Seong Woon Roh,
Young-Do Nam, and Jin-Woo Bae
Department of Life and Nanopharmaceutical Sciences and
Biology, Kyung Hee University
Haneul Kim*, Yochan Joung, and Kiseong Joh
Department of Bioscience and Biotechnology,
Hankuk University of Foreign Studies
A novel, Gram-positive, aerobic, rod-shaped, and non-motile
bacterial strain NAL101T was isolated from gajami-sikhae, a
traditional Korean fermented seafood made with flatfish.
Growth occurs at 4-45°C, pH 5-10, 0-12% (w/v) NaCl.
Optimum growth occurs at 30-37°C, pH 8, 0-1% NaCl. The
cell-wall amino acids were 2,4-diaminobutyric acid, alanine,
glycine, threonine and glutamic acid and the major fatty acid
components were anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The
16S rRNA gene sequence similarity value of strain NAL101T
and Leucobacter chironomi DSM 19883, most closely related,
is 97.7%. The DNA G+C content was 68.8 mol% and the
highest DNA-DNA hybridization values were 22.2%. Phylogenetic analysis based on 16S rRNA gene sequence, physiological, and biochemical characteristics indicated that the strain
NAL101T represents a new species of the Leucobacter (family
Microbacteriaceae), for which we propose the name Leucobacter celer sp. nov.. The type strain is NAL101T (=KACC
14220T= JCM 16465T).
A non-motile, Gram-staining-negative, yellow pigmented, rodshaped, strictly aerobic bacterium, strain HMD3159T, was
isolated from a saltern in Korea. The major fatty acids were
iso-C15:0(26.3%), iso-C17:0 3OH(12.1%), iso-C16:0(12.0%),
Summed Feature 3(comprising C16:1 ω7c and/or C16:1 ω6c ;
11.0%) and Summed Feature 9(iso-C17:1 ω9c and/or 10-methyl
C16:0; 10.0%). The major respiratory quinone was MK-6. The
DNA G+C content was 40.9 mol%. Phylogenetic tree based on
16S rRNA gene sequence showed that strain HMD3159T
formed a lineage within the genus Gramella and was closely
related to Gramella portivictoriae(94.9% sequence similarity),
Gramella echinicola(94.6%) and Gramella marina(93.6%).
On the basis of the evidence presented in this study, strain
HMD3159T represent a novel species of the genus Gramella,
for which the name Gramella jeungdonese sp. nov., is
proposed the type strain HMD3159T (=KCTC 23123T= CECT
ingT).
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A005
Leucobacter salsicius sp. nov. from a Salt-Fermented
Food
Ji-Hyun Yun1*, Seong Woon Roh1,2, Min-Soo Kim1,2,
Mi-Ja Jung1, Eun-Jin Park1, Kee-Sun Shin2,
Young-Do Nam1, and Jin-Woo Bae1
1
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University, 2University of
Science and Technology, Korea Research Institute of Bioscience
and Biotechnology
Strain M1-8T was isolated from a Korean fermented food. It
exhibited optimal growth between 25-30°C at pH 7.0-8.0 and
in 0-4% (w/v) NaCl. It can tolerate up to 10.0 mM Cr (VI).
Phylogenetic analyses of 16S rRNA sequences indicated that
strain M1-8T represents a new species in the genus Leucobacter.
The 16S rRNA gene sequence of M1-8T exhibits 98.1%
homology to that of Leucobacter chromiireducens subsp.
chromiireducens L-1T. The chromosomal G+C content of strain
M1-8T was 62.8 mol%. Its cell wall peptidoglycan contained
DAB, glutamic acid, alanine, glycine and GABA. The major
menaquinone was MK-11 and the predominant fatty acids were
anteiso-C15:0 (63.6%), anteiso-C17:0 (16.7%) and iso-C16:0 (14.2%).
The polar lipid profile of strain M1-8T contained diphosphatidylglycerol and one unknown glycolpid. Significant genotypic
and phenotypic differences were found between strain M1-8T
and other Leucobacter species. These differentiating characteristic indicate that strain M1-8T (= KACC 21127T= JCM
16362T) represents a novel species of the genus Leucobacter,
for which the name Leucobacter salsicius is proposed.
A007
Lentibacillus jeotgali sp. nov., a New Halophilic
Bacterium Isolated from Traditional Korean
Fermented Seafood
Mi-Ja Jung*, Seong Woon Roh, Min-Soo Kim, and
Jin-Woo Bae
Department of Biology, Kyung Hee University
A novel, Gram-positive, non-motile, endospore-forming and
moderately halophilic bacterium, strain GrbiT, was isolated
from a traditional Korean fermented seafood. The organism
grew optimally in the presence of 10-15% NaCl, at 37°C and
pH 8.0. The peptidoglycan of the cell wall consisted of mesodiaminopimelic acid, and the predominant menaquinone was
MK-7. The major fatty acids of strain GrbiT were iso-C16:0
(36.4%), anteiso-C15:0 (30.3%) and iso-C14:0 (18.2%). The polar
lipids were phosphatidylglycerol, diphosphatidylglycerol and
an unidentified glycolipid. The genomic DNA G+C content
was 42.5 mol%. Strain GrbiT was most closely related to the
type strain Lentibacillus kapialis JCM 12580T, with which it
shared 97.5% 16S rRNA gene sequence similarity. The DNADNA hybridization value between strains GrbiT and L. kapialis
JCM 12580T was 8%. Based on phenotypic, genotypic and
phylogenetic data, strain GrbiT should be classified as a novel
species within the genus Lentibacillus, for which the name
Lentibacillus jeotgali sp. nov. is proposed. The type strain is
GrbiT (= KCTC 13300T = JCM 15795T).
[Supported by TDPAF]
[Supported by grants from the TDPAF]
A006
A008
Microbacterium mitrae sp. nov., Isolated from Salted
Turban Shell
Oceanobacillus kimchii sp. nov. Isolated from a
Traditional Korean Fermented Food
Yun-Ji Kim*, Seong Woon Roh, Mi-Ja Jung, Min-Soo Kim,
Eun-Jin Park, and Jin-Woo Bae
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University
Tae Woong Whon1*, Mi-Ja Jung1, Seong Woon Roh1,2,
Young-Do Nam1, Eun-Jin Park1, Kee-Sun Shin2, and
Jin-Woo Bae1
1
Department of Life and Nanopharmaceutical Sciences, and
Department of Biology, Kyung Hee University, 2University of
Science and Technology, Korea Research Institute of Bioscience
and Biotechnology
A novel bacterium (strain M4-8T) belonging to the genus
Microbacterium was isolated from salted turban shell, which is
a traditional fermented food in Korea. The cells of this strain
were Gram-positive, non-motile, non-spore-forming rodshapes. The optimal growth condition was 1% (w/v) NaCl and
30°C. Phylogenetic analysis based on the 16S rRNA gene
sequences. Within the phylogenetic tree, this novel strain
shares a branching point with species Microbacterium hominis
IFO 15708T (97.8%). G+C content was 71.3 mol%, and DNADNA hybridization experiments showed a low level (<29%) of
DNA-DNA relatedness between M4-8T and its closest relatives.
The major fatty acids were C15 : 0 iso and C15 : 0 anteiso and
major cell-wall diamino acid is ornithine. Data obtained from
DNA–DNA hybridization and chemotaxonomic phenotypic
analysis supports the conclusion that strain M4-8T represents a
novel species within the genus Microbacterium, and we would
like to propose the name Microbacterium mitrae sp. The type
strain is M4-8T (= KACC 21129T = JCM 16363T).
[Supported by grants from the TDPAF (Technology
Development Program for Agriculture and Forestry)]
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Poster Sessions
The Gram-positive strain X50T was isolated from mustard
kimchi, a traditional Korean fermented food. The organism
grew under conditions ranging from 0-15.0% (w/v) NaCl
(optimal: 1-5%), 10-37°C (optimal: 25-30°C), and pH 5.3-9.3
(optimal: pH 7.5). Phylogenetic analysis based on the 16S
rRNA gene sequence indicated that strain X50T belongs to the
genus Oceanobacillus and is closely related phylogenetically
to the type strain of O. iheyensis HTE831T (98.9%) and O.
oncorhynchi subsp. Oncorhynchi 20AGT (97.0%). The cellular
fatty acid profiles predominately included anteiso-C15 : 0, and
iso-C15 : 0. The G+C content of the genomic DNA of the type
strain was 37.9 mol%, and the major isoprenoid quinone was
MK-7. Analysis of the 16S rRNA gene sequences, DNA-DNA
relatedness, and physiological and biochemical tests indicated
genotypic and phenotypic differences between strain X50T and
reference species in the genus Oceanobacillus. Therefore,
strain X50T was proposed as a novel species and named
Oceanobacillus kimchii. The type strain of the new species is
X50T.
A009
Phycicoccus sp. Isolated from Soil of a Potato
Cultivating Field
Hyangmi Kim1,2*, Doo-Sang Park1, Hyun-Woo Oh1,
Kang Hyun Lee1, Hee-Moon Park2, and Kyung Sook Bae1
1
Biological Resources Center, Korea Research Institute of
Bioscience and Biotechnology, 2Department of Microbiology,
Chungnam National University
Two Gram-positive, motile, cocci bacterium, strains L1b-b9T
and B5a-b5, were isolated from a potato cultivating field in
Ochang. Strain L1b-b9T grew at 10-45°C, pH5.0-10.0 and in
the presence of 8%(w/v) NaCl. Major menaquinone was MK8(H4) and the main cellular fatty acids were iso-C14:0, iso-C15:0
and anteiso-C15:0. Polar lipids in strain L1b-b9T consisted of
diphosphatidylglycerol and phosphatidylglycerol. The G+C
content of genomic DNA was 73.6 mol%(HPLC). Phylogenetic
analysis based on 16S rRNA gene sequences showed that
strains L1b-b9T and B5a-b5 shared 99.36% similarity and
formed a robust clade with the type species of the genus
Phycicoccus. Strains L1b-b9T and B5a-b5 were related most
closely to Phycicoccus aerophilus 5516T-20 (97.13% 16S
rRNA gene sequence similarity). The DNA-DNA relatedness
values between the two novel isolates and Phycicoccus
aerophilus (KACC20658) were below 70%.
A010
Bifidobacterium stercoris sp. nov., Isolated from
Human Faeces
Min-Soo Kim1,2*, Seong Woon Roh1,2, and Jin-Woo Bae1,2
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University, 2University of
Science & Technology, Biological Resources Center, Korea
Research Institute of Bioscience and Biotechnology
1
Strain Eg1T, an anaerobic, Gram-positive, non-motile and nonspore-forming bacterium, was isolated from human faeces.
F6PPK activity was positive. The end products of glucose
fermentation were acetic acid and lactic acid, which were
produced at a molar ratio of 1.76 : 1. The G+C content was
57.8 mol%. Comparative analyses based on the 16S rRNA and
hsp60 gene sequences showed that the isolate was closely
related to two species of the genus Bifidobacterium: B.
adolescentis (98.36% and 99.35% sequence homology, respectively) and B. ruminantium (97.93% and 92.13% sequence
homology, respectively). Despite this degree of sequence
similarity being high enough for the isolate to be included at
the same species level as B. adolescentis and B. ruminantium
(96.5–100%), low level of DNA-DNA relatedness (41%)
indicated that the isolate was distinguishable from these related
strains, B. adolescentis and B. ruminantium. Based on
phenotypic, genotypic and phylogenetic analyses, we propose
that the isolate from human faeces be classified as Bifidobacterium stercoris sp. nov., and designated as strain Eg1T (=
KCTC 5756T = JCM 15918T).
[Supported by grants from KFDA]
A011
Leifsonia moechotypicola sp. nov., a Xylanolytic
Bacterium Isolated from the Gut of Hairy LongHorned Toad Beetle Moechotypa diphysis (Pascoe)
Byung-Chun Kim*, Doo-Sang Park, Hyangmi Kim,
Hyun-Woo Oh, Kang Hyun Lee, Kee-Sun Shin, and
Kyung Sook Bae
Microbial Resources Center, Korea Research Institute of
Bioscience and Biotechnology
A novel Gram-positive, non-motile, rod-shaped bacteria,
designated strain RB-62T, was isolated during the study of
culturable bacteria in the gut of Moechotypa diphysis (Pascoe).
This isolates grew at 15-30°C and pH 5.0-7.5. The predominant isoprenoid quinone was MK-11, and the major
components of its cellular fatty acids were anteiso-C15 : 0
(44.1%), anteiso-C17:0 (23.3%), and iso-C16:0 (20.3%). The
DNA G+C content of the genomic DNA was 70.2 mol%
(HPLC). Phylogenetic analysis based on 16S rRNA gene
sequences showed that strain RB-62T was affiliated with a
cluster within the family Microbacteriaceae. The type strain
that was most closely related to strain RB-62T was Leifsonia
pindariensis PON10T (97.70%) and then Leifsonia ginsengi
wged11T (97.63%). The DNA–DNA relatedness value among
strain RB-62T, L. pindariensis PON10T and L. ginsengi
wged11T was under 70%. The phenotypic and phyogenetic
characteristics of the isolates allowed this isolate to be clearly
distinguished from other Leifsonia species. Based on these
polyphasic data, the strain RB-62T is considered to represent a
novel taxon of the genus Leifsonia, for which the name
Leifsonia moechotypicola sp. nov. is proposed.
A012
Phylogenetic Diversity and Abundance of the
Intestinal Bacterial Community in the Food Waste
Reducing Larvae of Hermetia illucens
Hyun Bum Jeon1*, So Young Park1, Jiyoung Choi2,
Gil-Sang Jeong2, Jonggill Kim2, Youngcheol Choi2, and
Sung-Jae Lee1
1
Department of Biology, College of Science, Kyung Hee University,
2
Laboratory of Environmental Entomology, National Academy of
Agricultural Science, Rural Development Administration
As it is discovered that food waste can be reduced by larval
Hermetia illucens, the scientific and commercial value of
larval BSF is increased recently. We extracted their intestinal
metagenomic DNAs from each larva provided with rice, calf
forage and food waste. We performed PCR by using bacterial
16S rRNA primers and pyrosequencing for the analysis of their
intestinal microbial community. We got the useful data set of
9744, 9768, and 5986 sequences from the PCR products of
food waste, rice, and calf forage fed samples, respectively. On
the basis of the BLAST search of EzTaxon program, the
bacterial community of the food waste fed gut was revealed
that the phyrum Bacteroidetes had dominancy of 67.31%,
Proteobacteria of 18.83%, and Firmicutes of 9.39%. The
phylogenic groups from rice fed gut were two dominant
phyrums of Firmicutes (42.09%) and Proteobacteria (53.77%).
The phylogenic groups from calf forage fed gut were four
similar portions of Firmicutes (23.39%), Actinobacteria
(24.69%), Bacteroidetes (20.51%), and Proteobacteria (30.91%).
We identified several organic compound degrading bacterial
strains: Bacillus amyloliquefaciens, Paenibacillus xylanedens,
Proteus mirabilis.
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A013
A015
Saccharospirillum aestuarii sp. nov., Isolated from
Tidal Flat Sedimentin the Yellow Sea
The Identification of Flavobacterium candidumensis
KUDC1076 sp. nov., Isolated from Dokdo Island
Ahyoung Choi*, Hyun-Myung Oh, and Jang-Cheon Cho
Inha University
Jong Myong Park1*, Seon-Ae Jeon1, Hye-Ri Sung1,
Jung-Hoon Yoon2, and Sa-Youl Ghim1
1
School of Life Sciences, Kyungpook National University,
2
Korea Research Institute of Bioscience and Biotechnology
A Gram-reaction-negative, non-motile, facultatively anaerobic
and curved rod-shaped bacterial strain, IMCC4453T, was
isolated from tidal flat sediment and subjected to a polyphasic
taxonomic study. Phylogenetic analysis based on 16S rRNA
gene sequences revealed that strain IMCC4453T belonged to
the genus Saccharospirillum. DNA-DNA relatedness between
strain IMCC4453T and S. salsuginis YIM-Y25T was 4.2%. The
DNA G+C content of the strain was 56.5 mol%. The major
polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lyso-phosphatidylethanolamine and the major isoprenoid quinone was Q-8.
Several physiological and biochemical characteristics between
strain IMCC4453T and the two Saccharospirillum species,
together with phylogenetic and genomic distinctiveness,
differentiated the strain from members of the genus Saccharospirillum. On the basis of polyphasic data obtained in this
study, it is concluded that strain IMCC4453T represents a novel
species of the genus Saccharospirillum, for which the name
Saccharospirillum aestuarii sp. nov. is proposed. The type
strain is IMCC4453T (KCTC 22684T = NBRC 105825 T).
A014
Optimization of Hexanoic Acid Production by
Megasphaera sp. nov. BS-4
Byoung Seung Jeon*, Seil Kim, Youngsoon Um, and
Byoung-in Sang
Korea Institute of Science and Technology
A new hexanoic acid-producing bacterium was isolated from
cow rumen using RCM(reinforced clostridia medium) with
hexanoic acid for isolation. The strain was designated
Megasphaera BS-4 based on 16s rDNA sequence and
phylogenetic tree. Physiological characteristics of the strain
were investigated using API 50 CH and batch fermentation.
Modified PYF medium was selected for cultivation of the
strain. Megasphaera BS-4 used fructose as the carbon source
and produced acetic acid, butyric acid, and hexanoic acid in
liquid phase and hydrogen in gas phase. The medium
composition for hexanoic acid production was optimized by
RSM (response surface methodology). When acetic acid and
butyric acid were added into the medium, concentration of
hexanoic acid increased up to 10 g/L in batch culture without
pH adjustment. The productivity of hexanoic acid was about
0.41 g/L/hr, which is higher than the result reported so far.
[Supported by grants from KETEP]
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Poster Sessions
Strain KUDC1076 was isolated from the rhizosphere of a
native plant Solanum nigrum L. in Dokdo island. Accorging to
transmission electron microscopic observation, KUDC1076
has peritrichous flagella, fimbriae, pili. It showed rod shape,
Gram negative and non spore forming. Phylogenetic analysis
based on 16s rRNA gene sequences showed that KUDC1076
belongs to genus Flavobacterium, being closest related with F.
johnsoniae (97.5%), F. anhuiense (96.9%), F. defluvii (96.4%)
and F. denitrificans (96.2%). KUDC1076 produces the
flexirubin type pigment which is a unique bacterial pigment of
Cytophaga and Flexibacter group and its related genera. On
the basis of the phenotypic and molecular data, strain
KUDC1076 represents a novel species within the genus
Flavobacterium, for which the name Flavobacterium candidumensis sp. nov. is proposed.
A016
The Identification of Paenibacillus taehanensis U5-6
sp. nov., Isolated from The Rhizosphere of Acer
okamotoanum, a Native Plant in Ulleungdo Island
Ye-Ji Hwang1*, Seon-Ae Jeon1, Hye-Ri Sung1,
Jung-Hoon Yoon2, and Sa-Youl Ghim1
1
School of Life Sciences, Kyungpook National University,
2
Korea Research Institute of Bioscience and Biotechnolgy
Strain U5-6 was isolated from the rhizosphere of Acer
okamotoanum, a native plant in Ulleungdo island. This strain
comprised Gram positive, endospore-forming, flagellar rods
and is capable of growth at 18~37°C with optimal growth
temperature of 30°C. This strain is oxidase- and catalasepositive, and does not hydrolyze starch and casein. The length
of this strain is from 2.0 to 2.5 μm. Phylogenetic analysis based
on 16s rRNA gene sequences showed that strain U5-6 belongs
to the genus Paenibacillus, being related to P. chondroitinus
(97.8%), P. alginolyticus (97.6%), P. pocheonesis (97.5%), P.
pectinilyticus (97.1%), and P. aestuarii (97.1%). On the basis
of the analysis of cellular fatty acid composition, G+C contents
and phenotypic data, strain U5-6 represents a novel species of
the genus Paenibacillus, for which the name Paenibacillus
taehanensis is proposed.
A017
A019
Inferring Bacterial Species Tree Using Multilocus
Sequence Data
Genotyping of Bacillus anthracis Korean Isolates
Using Molecular Typing Methods
Mincheol Kim* and Jongsik Chun
School of Biological Sciences and Institute of Microbiology,
Seoul National University
Sang Hoon Kim*, Sudipto Shahid, Kyoung Hwa Jung,
Dong In Jeon, and Young Gyu Chai
Division of Molecular and Life Sciences, Hanyang University
Inferring species tree in bacteria is of intense interest because
there is no clear species definition in bacteria and bacterial
molecular phylogeny has relied mainly on 16S rRNA gene
which is known for the lack of consistency over the whole
bacterial taxa. Recently, the combined information from
multiple genes has been used as information for inferring
bacterial species trees. The method concatenating sequences
from multiple genes has been widely used, but it frequently
leads to poor estimation of the species trees. Several alternative
approaches, such as Bayesian approach and consensus and so
on, have recently been tried to infer species trees accurately
without biases. Here we tested the applicability of several
promising tools, which are based on multispecies coalescent,
into multilocus sequence data of the gut symbiont
Lactobacillus group. The result showed that bacterial species
tree was estimated more precisely and accurately by the
multispecies coalescent based methods compared to the
concatenation method. The development of a new approach
regarding horizontal gene transfer and recombination with
coalescence is warranted for following study.
Bacillus anthracis is the bacterium that causes fetal anthrax to
a wide range of herbivores and even humans is closely related
and often critical to differentiate from other members of the
Bacillus cereus group that can causes diverse diseases.
Identification and detection of these threatening bugs through
molecular approaches is enhancing the insights in to the
process of microbial population genetics and pathogenesis of
infectious diseases. In our study we have employed several
molecular approaches like single-nucleotide polymorphisms
analysis, multilocus variable number tandem repeat analysis
and single–nucleotide repeats for the molecular typing of B.
anthracis Korean isolates. Using those efficient genotyping
methods we screened the genetic diversity and have found
highly similar with two major lineages (A and B) for the first
time of those isolates. Furthermore, results suggest a greater
understanding of the genetic diversity of B. anthracis in natural
populations may be required to definitively distinguish a
natural outbreak from an intentional use situation.
A018
A020
Isolation of a New Alcohol-Producing Clostridium
Species from Tidal Flat
Chitiniphilus sp. nov., a Novel Chitin-Degrading
Bacterium Isolated from Farming Field
Kieun Choi1,2*, Byoung Seung Jeon1, Seil Kim1,
Min-Kyu OH2, Youngsoon Um1, and Byoung-In Sang1
1
Korea Institute of Science and Technology, 2Department of
Chemical and Biological Engineering, Korea University
Jae-Chan Lee*, Dong-Jin Park, and Chang-Jin Kim
Korea Research Institute of Bioscience and Biotechnology
Butanol and ethanol are alternative biofuels for transportation.
The bioproduction of butanol and ethanol is generally found in
cultures of solventogenic Clostridium species through acetonebutanol-ethanol (ABE) fermentation. For the isolation of new
alcohol-producing Clostridium species, tidal flat samples were
collected from seashore area of Tae-An, Korea. The isolated
bacterium was designated Clostridium sp. nov. BS-5 and its
16S rDNA revealed 96% similarity to Clostirdium algidixylanolyticum SPL73T. Products of Clostridium sp. nov. BS-5
were acetone, ethanol, butanol, acetic acid, and butyric acid
from glucose as a carbon source. To improve alcohol
production, P2 medium supplemented with 3% sea salt and 5
g/L yeast extract was used and sodium butyrate was initially
added 5g/L to investigate butanol production. Optimized
temperature for alcohol production was 30°C. At the optimized
conditions, Clostridium sp. nov. BS-5 produced 26.1 g/L
alcohol with the yield of 0.43 g/L. This result shows that
Clostridium sp. nov. BS-5 is a promising new microorganism
for alcohol production.
Chitiniphilus sp. nov. is a bacterial strain capable of degrading
chitin isolated from farming field in Daejeon, Korea. The strain
was Gram-negative rod-shaped facultatively anaerobic and
motile with a single flagellum. It grew well with chitin as a
single carbon source. It grows best at 0-7% w/v NaCl. The
predominant respiratory lipoquinone found in the strain is
ubiquinone with eight isoprene units. Phylogenetic analyses
strongly indicate that this strain forms a distinct line within a
clade containing the genus Chitiniphilus and the closest
similarity value to Chitiniphilus shinanonensis SAY3T with
95.9%. On the basis of the polyphasic evidence gathered in this
study it is proposed that the isolate is a new species, Chitiniphilus nov. sp.
[Supported by the Microbial Genomes & Application Center
of 21st Century R&D Program, MEST]
[Supported by grants from KEMCO]
139
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A021
A023
Identification and Characterization of Paenibacillus
sp. nov., Isolated from Farming Field in Korea
*
Jae-Chan Lee , Dong-Jin Park, and Chang-Jin Kim
Korea Research Institute of Bioscience and Biotechnology
A Gram positive, non-motile, rod-shaped bacterium, strain
BH038, was isolated from a farming field in Korea. Strain
BH038 grew optimally at 30-33°C, pH 7·2-7·5 and 0% (w/v)
of NaCl. The isolate showed oxidase- and catalase-negaitive
reactions and did not reduced nitrate to nitrite. Anaerobic
growth did not occur on TSA agar (Difco). The major cellular
fatty acids were anteiso-C15:0, iso-C16:0 and iso-C15:0. The levels
of 16S rRNA sequence similarity of strain BH038T to
Paenibacillus chibensis JCM 9905T, Paenibacillus barengoltzii
DSAFN-016T, Paenibacillus timonensis 2301032T, Paenibacillus chibensis NRRL B-142T, Paenibacillus motobuensis
MC10T, Paenibacillus konsidensis LBYT were 96.3%, 96.0%,
95.9%, 95.5%, 95.5% and 95.3%, respectively. On the basis of
combined phenotypic properties and phylogenetic and genetic
data, strain BH038 should be placed in the genus Paenibacillus
as the type strain of a novel species.
Additions to Leprarioid Lichens in South Korea
Yogesh Joshi*, Young Jin Koh, and Jae-Seoun Hur
Korean Lichen Research Institute, Sunchon National University
The presentation contributes to the study of new records of
leprarioid lichens reported for the first time from this country
as well as the species earlier reported from this part of the
continent and expands the knowledge of lichen diversity in
East Asia including China and Japan. Twenty taxa belonging
to four different genera Chrysothrix, Lepraria, Lepraucaulon
and Normandina respectively have been reported out. Except
Lepraria coriensis and Normandina pulchella, all other species
described here are new to South Korea. Among them, Lepraria
caesiella, L. eburnea, L. leprolomopsis, L. lobata, L. pallida, L.
texta and L. xerophila are reported for the first time in East
Asia including China and Japan. Brief taxonomic description
and comments of each species are provided.
[Supported by the Microbial Genomes & Application Center
of 21st Century R&D Program, MEST]
A022
A024
A Novel Ornithinicoccus sp. nov., Isolated from
Bigeum Island in Korea
*
Jae-Chan Lee , Dong-Jin Park, and Chang-Jin Kim
Korea Research Institute of Bioscience and Biotechnology
A Gram-positive coccoid, non-motile bacteria with L-ornithine
as diagnostic diamino acid of the peptidoglycan were isolated
from a sample of farming field of Bigeum island in Korea.
This strain grew optimally at 28-33°C, pH 6.8-7.5 and 3%
(w/v) of NaCl. This strain forms a distinct line within a clade
the genus Ornithinicoccus and the cloestest type strain was
Ornithinicoccus hortensis KHI 0125T with the similarity of
96.1%. The other close type strains to the strain were
Oryzihumus leptocrescens KV-628T, Lapillicoccus jejuensis RAc013T, Konellia aerolata 5317S-21T and Tetrasphaera
duodecadis ATCC 5116T with the sequence simililarity of
95.6%, 95.5%, 95.0%, and 94.9% respectively. On the basis of
combined phenotypic properties and phylogenetic and genetic
data, this strain should be placed in the genus Ornithinicoccus
as the type strain of a novel species.
[Supported by the Microbial Genomes & Application Center
of 21st Century R&D Program, MEST]
140
Poster Sessions
Additions to Foliicolous Lichen Flora of Vietnam
Thi Thuy Nguyen1*, Yogesh Joshi1, Anh Dung Nguyen2,
Young Jin Koh1, and Jae-Seoun Hur1
1
Korean Lichen Research Institute, Sunchon National University,
2
Plant Biological Department, Faculty of Agriculture, Tay
Nguyen University, Vietnam
Foliicolous lichens (i.e. lichens growing over leaves) have
been well worked out in Neotropics, while fewer data are
available from tropical Africa and SE Asia. Fewer publications
have come out from Vietnam mentioning the occurrence of 70
foliicolous taxa from this place. The authors describes six
additional new records of foliicolous lichens from Vietnam
belonging to three different families-Gomphillaceae (Aderkomyces albostrigosus fo. albostrigosus, A. albsostrigosus fo.
aggregatus and Asterotherium rotuliforme), Pilocarpaceae
(Byssoloma vanderystii and Fellhanera emarginata) and
Porinaceae (Trichothelium minutum), thus adding to the
knowledge of the Vietnam lichen biota and increasing the tally
of foliicolous lichens to 76. The specimens were collected at
Thác Dray Sap (commonly called Dray Sac Waterfall) region
situated in the Central Highland part of Vietnam. Most of the
specimens were collected from understory leaves where high
humidity and low light intensity prevail. This number
represents only a small fraction of the actual foliicolous lichen
diversity in the country, and further studies in this area will
definitely increase the tally.
A025
A027
Tenacibaculum luteolum sp. nov., Isolated from a
Marine Sponge
Sphingopyxis soli sp. nov., Isolated from Landfill
Soil
Sung-Hyun Yang1*, Olga I. Nedashkovskaya2,
Hyun-Seok Seo1, Sung Hyuk Lee1, Jung-Hyun Lee1,
Sang-Jin Kim1, and Kae Kyoung Kwon1
1
Marine Biotechnology Research Center, Korea Ocean Research &
Development Institute, 2Pacific Institute of Bioorganic Chemistry of
the Far-Eastern Branch of the Russian Academy of Sciences, Russia
Jung-Hye Choi1*, Min-Soo Kim1,2, Mi-Ja Jung1,
Seong Woon Roh1,2, Kee-Sun Shin2, and Jin-Woo Bae1
1
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University, 2University of
Science & Technology, BRC, Korea Research Institute of
Bioscience and Biotechnology
A novel psychrotrophic bacterium, strain HJ103T, was isolated
from a marine sponge collected in the East Sea, Korea (also
known as Sea of Japan). Cells were Gram-negative, motile by
gilding, strictly aerobic, catalase-, and oxidase-positive, rodshaped (0.3-0.5 μm×1.6-6.2 μm) and halophilic. Growth was
observed between 5 and 33°C (optimum 26°C), at pH 6.5-9.0
(optimum 8.0) and in the presence of 2.0-3.5% (optimum
2.5%) NaCl. The strain requires Ca2+, Mg2+ and K+ ions for
growth. The 16S rRNA gene sequence analysis revealed that
strain HJ103T showed high similarity with members of the
genus Tenacibaculum (94.4-96.4%), and phylogenetic analysis
revealed that strain HJ103T shared a phyletic line with Tenacibaculum maritimum. The major respiratory quinone is MK-6.
The DNA G+C content was 31.1 mol%. The major fatty acids
were iso-C15:0, iso-C15:0 3-OH, C16:0 3-OH and summed feature
3 comprising C16:1 ω7c and/or iso-C15:0 2-OH. On the basis of
this polyphasic taxonomic evidence, strain HJ103T should be
classified as a species in the genus Tenacibaculum and the
name as Tenacibaculum luteolum sp. nov. is proposed. The
type strain is HJ103T (= KCCM 42328T = JCM 14618T).
Yellow pigmented bacterium (on LB agar) plate was isolated
from landfill soil in Pohang, Republic of Korea and designated
as BL03T. Strain BL03T is Gram-negative, aerobic, rod-shaped,
motile, oxidase- positive and catalase- negative. Temperature
range of the strain is 15 to 42°C, and pH range is 5.0 to 9.5.
Growth occurs at 0 to 3% (w/v) NaCl. Predominant ubiquinone
of the strain characterized chemotaxonomically as containing
Q-10 and the major cellular fatty acids were C17:1ω6c, C15:0
2OH and C18:1ω7c. Phylogenetic analysis based on 16S rRNA
gene sequence showed that strain BL03T belongs to the genus
Sphingopyxis, showing high sequence similarity to the type
strain Sphingopyxis taejonensis JSS54T (97.8%), Sphingopyxis
alaskensis RB2256T (97.4%) and Sphingopyxis chilensis S37T
(96.9%). The GC content of the DNA strain was 65.9 mol%.
Several characteristics served to differentiate this isolate from
recognized members of the genus Sphingopyxis. Strain BL03T
(KCTC 22405T = JCM 15910T) should be classified as novel
species in the genus Sphingopyxis, which the name Sphingopyxis soli sp. nov., is proposed.
[Supported by MEGRC]
A026
Agromyces atrinae
Fermented Seafood
[Supported by grant from the Eco-technopia 21]
A028
sp.
nov.,
Isolated
from
Screening and Identification of Antibacterial Lactic
Acid Bacteria Isolated from Kimchi
Eun-Jin Park*, Min-Soo Kim, Mi-Ja Jung, Seong Woon Roh,
and Jin-Woo Bae
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University
Ji Eun An*, Hyoung Ro Lee, Ji Sun Kim, and Nam Soo Han
Department of Food Science and Technology, Chungbuk
National University
A Gram-positive, aerobic, non-motile and rod-shaped
bacterium, designated P27T, was isolated from a traditional
fermented seafood. The isolate grew optimally in 0–2.0%
(w/v) NaCl, pH 6–7, at 30°C. The predominant menaquinones
were MK-12 and MK-11. The major cellular fatty acids were
anteiso-C17:0, anteiso-C15:0 and iso-C16:0. The major cell wall
sugars were galactose, mannose and rhamnose. The peptideglycan amino acids of strain 27T were 2,4-diaminobutyric acid,
alanine, glutamic acid and glycine. The major polar lipids were
diphosphatidylglycerol, phosphatidylglycerol and an unidentified
glycolipid. The genomic DNA G+C content of strain P27T was
69.0 mol%. Based on its 16S rRNA gene sequence, strain P27T
showed highest pairwise similarity with Agromyces cerinus
subsp. cerinus JCM 9083T with 97.0% similarity value. Based
on phenotypic, genotypic and phylogenetic studies, strain P27T
represents a novel species in the the genus Agromyces, for
which the name Agromyces atrinae sp. nov. is proposed. The
type strain is P27T (=KCTC 19593T= JCM 15913T).
The purpose of this study was to screen and identify the
antibacterial lactic acid bacteria from kimchi by using spot-onthe-lawn method. Listeria monocytogenes ATCC 19115,
Staphylococcus aureus KCTC 1916, Escherichia coli KCTC
1467 and Salmonella typhimurium KCTC 2515 were used as
indicators. Approximately 100 strains showed antimicrobial
activity inhibiting growth of indicators on MRS and PEA
media, and 21 strains were isolated by their cell morphology,
gram stain and high antimicrobial activity. The 21 isolates
were identified as Leuconostoc mesenteroides, Leuconostoc
pseudomesenteroides, Weissella cibaria, Lactococcus lactis
and Enterococcus faecalis by 16S rRNA gene sequencing.
[Supported by grant from TDPAF]
141
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A029
A031
Community Structure of Culturable Bacteria
Associated with Sponge, Penares incrustans
Mucilaginibacter dorajii sp. nov., a Novel Bacterium
Isolated from the Platycodon grandiflorum
Il-Gyo Jang* and Jin-Sook Park
Department of Biotechnology, Hannam University
Kee-Sun Shin*, Byung-Chun Kim, Kang Hyun Lee, and
Mi Na Kim
Biological Resource Center, Korea Research Institute of
Bioscience and Biotechnology
A cultivation-based approach was employed to determine the
culturable bacterial diversity associated with marine sponges,
Penares incrustans. The diversity of culturable bacteria
collected from seawater which the marine sponge is inhabiting,
was also analyzed as control. The bacteria associated with
sponge were cultivated using Zobell medium for 3 days in
25°C incubation. Community structures of the culturable
bacteria were analyzed with PCR-RFLP based on 16S rDNA
sequences. The sequence analysis of each RFLP type showed
>97% similarity to known bacterial species in public databases.
Overall, the microbial populations of Penares incrustans
sponges investigated were found to be the members of the
classes; Alphaproteobacteria (29.0%), Gammaproteobacteria
(64.5%), and Actinobacteria (6.5%). The dominant bacterial
group of seawater was also found to be the members of the
Gammaproteobacteria. But, the bacterial community of
seawater showed to be more simple than that of the sponge. In
bacterial species composition of Gammaproteobacteria, Vibrio
(43%) and Pseudoalteromonas (60.6%) were dominant in
Penares incrustans and seawater respectively.
A030
Community Analysis of Culturable
Associated with Sponge, Asteripus sp.
A032
Bacteria
Choon-Soo Im* and Jin-Sook Park
Department of Biotechnology, Hannam University
A cultivation-based approach was employed to determine the
culturable bacterial diversity associated with marine sponges,
Asteripus sp. The diversity of culturable bacteria was also
analyzed as control. The bacteria associated with sponge were
cultivated using Zobell medium for 3 days in 25°C incubation.
Community structures of the culturable bacteria were analyzed
with PCR-RFLP based on 16S rDNA sequences. The RFLP
fingerprinting of 16S rDNA digested with HaeIII and MspI,
revealed 12 and 8 independent RFLP types from the sponge
species and seawater respectively. The sequence analysis of
each RFLP type showed >96% similarity to known bacterial
species in public databases. Four other strains were potential
candidates for new species. The dominant microbial populations of Asteripus sp. were found to be the members of the
Alphaproteobacteria (78.5%). But, predominant microbial
group of seawater was Gammaproteobacteria (81.8%). The
bacterial community of the sponge and seawater indicated
similar composition at phyla level, revealing difference in
dominant bacterial species. MspI was more useful than HaeIII
in PCR-RFLP analyzing for bacterial diversity of marine
sponge, Asteripus sp.
142
Poster Sessions
A Gram-negative, yellow-pigmented, rod-shaped bacterial
strain, DR-f4, was isolated from the rhizosphere of Platycodon
grandiflorum in a study of bacterial diversity in the rhizosphere,
and its taxonomic position was investigated by a genotypic and
phenotypic analysis. It grew 10-30°C, optimally at 20-25°C,
and in the presence of 0–1% (w/v) NaCl. It contained MK-7 as
the predominant menaquinone. It represented positive activity
for catalase, oxidase, β-galactosidase and hydrolysis of esculin.
The major cellular fatty acids were iso-C15:0 2OH/C16:1ω7c and
iso-C15:0. The DNA G+C content was 42.6 mol%. This isolate
belonged to the genus Mucilaginibacter based on phylogenetic
analysis using 16S rRNA gene sequences. The nearest
phylogenetic
neighbours
of
strain
DR-f4T
were
T
Mucilaginibacter lappiensis ANJL12 and Mucilaginibacter
rigui WPCB113T, with 16S rRNA gene sequence similarity
levels of 96.9% and 96.4%, respectively. The genetic and
phenotypic evidences suggest that strain DR-f4T should be
classified as a novel species, for which the name Mucilaginibacter dorajii sp. nov. is proposed. The type strain for the
novel species is DR-f4T (=KACC 14556T = CECT 7660T).
Dietzia alimentaria sp. nov., Isolated from a
Traditional Korean Food
Jandi Kim*, Seong Woon Roh, Mi-Ja Jung, Min-Soo Kim,
Eun-Jin Park, and Jin-Woo Bae
Department of Biology, Kyung Hee University
An actinobacterial strain 72T was isolated from a traditional
salt-fermented seafood in Korea. Colonies were coral-red, and
cells were Gram-positive, non-motile and rod shaped. Strain
72T grew in 0–10% (w/v) NaCl, at pH 7–10 and a temperature
of 15-37˚C. Optimum growth conditions were 2% NaCl, pH
7.0 and 30˚C. Phylogenetic analysis indicated that strain 72T
belonged to the genus Dietzia. The major cellular fatty acids
were C16:0 (33.2%) and C18:1 ω9c (17.1%), both of which are
characteristic of members of the genus Dietzia. Analysis of the
16S rRNA gene sequences and DNA-DNA hybridization,
coupled with physiological and biochemical tests, determined
genotypic and phenotypic differences between strain 72T and
other members of the genus. Based on these data, strain 72T is
a novel species of the genus Dietzia, and we propose the name
Dietzia alimentaria sp. nov. The type strain is 72T (= JCM
16360T = KACC 21126T).
A033
Algoriphagus jejuensis, sp. nov., Isolated from
Seawater
Ki Young Lee1*, Dong-Heon Lee1, Hyung-Yeel Kahng2, and
Sun Bok Lee1,3
1
Department of Chemical Engineering, Pohang University of
Science and Technology, 2Department of Environmental Education,
Sunchon National University, 3Gyungbuk Sea Grant Institute,
Pohang University of Science and Technology
A Gram-negative, pink-pigmented, non-motile, strictly aerobic,
rod-shaped bacterium, designated CNU040T, was isolated from
seawater on the coast of Jeju Island in Korea, and subjected to
a polyphasic taxonomic study. Phylogenetic analysis based on
16S rRNA gene sequences indicated that strain CNU040T
belongs to a distinct lineage in the Algoriphagus, with high
sequence similarity to Algoriphagus terrigena DS-44T (98.3%).
DNA-DNA relatedness values between the strain CNU040T
and Algoriphagus terrigena KCTC 12454T were 44.5%. The
DNA G+C content of the strain was 48.5 mol% and the major
respiratory quinone was menaquinone-7. The major cellular
fatty acids were iso-C15 : 0 (28.6%), summed feature 3 (iso-C15:0
2-OH/C16:1ω7c, 24.0%) and iso-C17:0 3-OH (4.7%). On the
basis of phenotypic, phylogenetic and genotypic data, strain
CNU040T represents a novel species within the genus
Algoriphagus, for which the name Algoriphagus jejuensis sp.
nov. is proposed. The type strain is CNU040T (=KCTC 22647T
= JCM 16112T ).
[This work was supported by the 21C Frontier Microbial
Genomics and Applications Centre Program, Ministry of
Education, Science & Technology, Korea].
A034
A035
Kopria litus gen. nov., sp. nov., of the Family
Oxalobacteraceae, Isolated from Antarctic Coastal
Seawater
Eun Hye Kim1,2*, Hyun-Jeong Jeong1, Yoo Kyoung Lee1,
Eun Young Moon3, Jang-Cheon Cho2, Soon Gyu Hong1,
and Hong Kum Lee1
1
Polar BioCenter, Korea Polar Research Institute, Korea Ocean
Research & Development Institute, 2Division of Biology and
Ocean Sciences, Inha University, 3Institute of Microbiology,
Seoul National University
A Gram-negative, non-motile, catalase- and oxidase- positive,
strictly aerobic and short-rod-shaped bacterium that was
designated strain KOPRI 25157T was isolated from coastal
seawater sample in around King Sejong Station, in King
George Island, Antarctica. The temperature and pH ranges for
growth on R2A agar were 10-20°C, and 5.0-10.0, respectively.
Phylogenetic analyses of the 16S rRNA gene sequence of
strain KOPRI 25157 T have shown that it belongs to the family
Oxalobacteraceae of the class Betaproteobacteria, but it
formed a distinct clade from other recognised members of the
family. Major ubiquinone was Q-8. Predominant cellular fatty
acids were C10:0 (0.2%), C10:0 3OH (3.2%), C12:0 (2.9%), C16:1
ω7c/15 iso 2OH (56.4%), C16:0 (30.5%), C18:1 ω7c (3.7%),
C18:0 (0.7%), and 11 methyl C18:1 ω7c (2.4%). On the basis of
these data, it is proposed that strain KOPRI 25157T is the
representative of a novel genus, named Kopria gen. nov. is
proposed in the family Oxalobacteriaceae. The type strain for
Kopria litus sp. nov. is KOPRI 25157T (=JCM 16673T =KCTC
23040 T).
A036
Notes on the Existence of Leucodecton desquamescens
(Thelotremoid Graphidaceae) in South Korea
Caenimonas terrae sp. nov., Isolated from a Soil
Sample in Korea
Yogesh Joshi*, Young Jin Koh, and Jae-Seoun Hur
Korean Lichen Research Institute, Sunchon National University
Soo-Jin Kim1*, Hang-Yeon Weon2, Yi-Seul Kim2,
Jung-Im Bok2, and Soon-Wo Kwon2
1
Korean Agricultural Culture Collection, Agro-biodiversity
Center, National Academy of Agricultural Science, Rural
Development Administration, 2Korean Agricultural Culture
Collection, Agro-biodiversity Center, National Academy of
Agricultural Science, Rural Development Administration
The presentation describes one new record of thelotremoid
lichen (Leucodecton desquamescens) from South Korea, which
is characterized by thick, bulging thallus with many calcium
oxalate crystal inclusions, the immersed, round to irregular
ascomata with free exciple, ellipsoid to ±roundish submuriform, brown ascospores and lack of secondary metabolites. A
detailed taxonomic description and comments are presented for
the taxa studied. Lichen genus Leucodecton is reported for the
first time for this country.
A white-coloured bacterium, SGM1-15T, was isolated from a
paddy soil sample from Suwon, Republic of Korea. The cells
were strictly aerobic, Gram-negative and curved rod-shaped
(0.6-0.7×1.4-2.3 μm). A phylogenetic analysis based on 16S
rRNA gene sequences revealed that strain SGM1-15T was
closely related to Curvibacter delicatus LMG 4328T (97.6%
similarity) and Caenimonas koreensis EMB320T (97.5%
similarity). The major respiratory quinone system was Q-8 and
the predominant cellular fatty acids were C16:0 (39.9%),
summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH; 24.3%)
and C17:0 cyclo (22.7%). The DNA G+C content was 68.7
mol%. On the basis of the phylogenetic, physiological and
chemotaxonomic data, stain SGM1-15T represents a novel
species of the genus Caenimonas, for which the name
Caenimonas terrae sp. nov. is proposed. The type strain of
Caenimonas terrae is SGM1-15 (=KACC 13365T=NBRC
106341T).
[Supported by grants from RDA]
143
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A037
A039
Cohnella soil sp. nov. and Cohnella terrae sp. nov.
Isolated from Two Different Soil Samples in Korea
Psychroserpens gangjinensis sp. nov., Isolated from
Seawater
Soo-Jin Kim*, Hang-Yeon Weon, Yi-Seul Kim,
In-Cheol Park, and Soon-Wo Kwon
Korean Agricultural Culture Collection, Agro-biodiversity
Center, National Academy of Agricultural Science, Rural
Development Administration
Young Sun Lee1*, San Ho Shon1, Jae Sung Oh1,
Dong-Heon Lee2, Hyung-Yeel Kahng2, and Jae Sung Jung1
1
Department of Biology, Sunchon National University,
2
Department of Environmental Education, Sunchon National
University
Two bacterial were isolated from soil samples in Korea, strains
YM2-7T and WD2-19T. The cells were strictly aerobic, Grampositive, motile with peritrichous flagella and rod-shaped.
YM2-7T and WD2-19T showed 16S rRNA gene sequence
similarities of 93.2-96.0% to type strains of recognized
Cohnella species. The G+C contents of the DNA of strains
YM2-7T and WD2-19T were 52.2 and 55.6 mol%, respectively.
Major fatty acids of strain YM2-7T are anteiso-C15:0 (44.4%),
C16:0 (19.2%) and iso-C16:0 (16.8%), and the major fatty acids
of strain WD2-19T are anteiso-C15:0 (46.5%), iso-C16:0 (21.8%)
and C16:0 (11.2%). Both strains contained MK-7 as the
predominant quinone. Both strains had diphosphatidylglycerol,
phosphatidylglycerol, phosphatidylethanolamine and lysylphosphatidylglycerol as the major polar lipids. Comparative
analysis of phenotypic and phylogenetic traits indicated that
strains YM2-7T and WD2-19T represented two novel species of
the genus Cohnella. The names Cohnella soli sp. nov. (type
strain YM2-7T = KACC 13346T = NBRC 106486T), and
Cohnella terrae sp. nov. (type strain WD2-19T = KACC
13347T= NBRC 106485T) are proposed for these organisms.
[Supported by grants from RDA]
Gram-negative, catalase and oxidase-positive, aerobic, yellow
pigmented strain, K7-7T, was isolated from sea water of the
Gangjin bay (34°32'58.0"N, 126°46'15.7"E) in Korea. Based
on 16S rRNA gene sequence comparisons, the novel strain was
closely related to genera Bizionia (96.192-93.626%), Psychroserpens (95.551-95.326%), and Winogradskyella (95.05693.922%) of the family Flavobacteriaceae. According to
Phylogenetic analyses, K7-7T 16S rRNA showed similarity
with Bizionia, but it was more distantly related with genera
Psychroserpens. It grew at 10-37°C (Optimum 30°C), at pH 78 and with 2-6% NaCl (Optimum 3%). Strain K7-7T was able
to hydrolysis Casein, Tween20, tween60, Tween80, DNase. It
is contained C15:0 ISO (31.2%), C15:1 ISO G (21.1%) and C17:0
ISO 3-OH (17.2%) as major fatty acid. The DNA G+C
contents was 37.48 mol%. On the basis of polyphasic
taxonomic data, strain K7-7T (=KCTC22734T = JCM16151T)
should be classified as the type strain of a novel species within
the genus Psychroserpens, for which the name Psychroserpens
gangjinensis sp. nov. is proposed.
A038
Thalassobacter haliotis sp. nov., Isolated from
Abalone (Haliotis discus)
Young Sun Lee1*, San Ho Shon1, Dong-Heon Lee2,
Hyung-Yeel Kahng2, and Jae Sung Jung1
1
Department of Biology, Sunchon National University,
2
Department of Environmental Education, Sunchon National
University
A rod-shaped, yellow-pigment, aerobic, Gram-negative
bacterium, strain JA14T, was isolated from the Haliotis discus
collected from sea of Wando-island(34°25'30.88"N, 126°56'
23.37"E), Korea. Phylogenetic analyses based on 16S rRNA
gene sequences revealed that strain JA14T shared 95.945%95.032% sequence similarity with species of the genus
Thalassobacter, and also Roseovarius (95.752-92.939%),
Pseudoruegerian (95.661-95.608%), Celeribacter neptunius
(95.326%) and Sulfitobacter (95.090-93.160%). However,
Phylogenetic analyses showed that it was most closely related
to genena Thalassobacter. It grew at 10-37°C (Optimum 25°C),
at pH 6-11 (Optimum 6) and with 1-4% NaCl (Optimum 1).
Strain JA14T was able to hydrolysis tween60. Major fatty acids
were C15:0 ISO (36.7%), C15:1 ISO G (12.4%), C17:0ISO 3-OH
(8.4%), C16:0 (6.2%), C15:0 ISO 3-OH (5.7%) and C15:0 ISO 2OH and/or C16:1 (5.7%). The DNA G+C contents was 28.74
mol%. These data, as well as phylogenetic analyses, suggest
that strain JA14T (=KCTC22732T =JCM16145T) should be
classified as the type strain of a novel species within the genus
Thalassobacter, for which the name Thalassobacter haliotis sp.
nov. is proposed.
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A040
Genetic Diversity of Pseudomonas syringae pv.
morsprunorum Strains Isolated from Prunus mume
and Relatedness with Other Pathovars Based on the
Comparative Sequence Analysis of the 16S rRNA
Gene
Young Sun Lee1*, Young Jin Koh2, and Jae Sung Jung1
1
Department of Biology, Sunchon National University,
2
Department of Plant Medicine, Sunchon National University
Genetic diversity among Pseudomonas syringae pv.
morsprunorum strains isolated from Prunus mume in Korea
and Japan was investigated by comparative sequence analysis
of the 16S rRNA gene. The strains included 24 field isolates
recovered in Korea and 7 Japanese isolates. Two strains
isolated from P. salicina in Japan, one strain from P. avium in
United Kingdom and the pathovar reference strain isolated
from Prunus domestica were also used for comparison of 16S
rRNA gene sequence. A nearly complete sequences of the 16S
rRNA genes were sequenced in 35 strains, and three types of
sequence, designated Group I, II and III, were identified. The
pathovar reference strain and 19 Korean strains belonged to
Group I, while 5 Korean, 5 Japanese strains and one strain
from United Kingdom belonged to Group II. Another 4
Japanese strains belonged to Group III. Five Korean strains
belonging to Group II were isolated from only five among 24
orchards investigated. When the nucleotide sequences of the
16S rRNA genes of P. syringae pathovars were aligned, the
sequence polymorphism was found to be focused on the
variable regions, which were shown in pv. morsprunorum
population.
A041
Reclassification of Paenibacillus ginsengisoli as a
Later Heterotypic Synonym of Paenibacillus
anaericanus
Kwang Kyu Kim*, Keun Chul Lee, and Jung-Sook Lee
Korean Collection for Type Cultures, Biological Resource
Center, Korea Research Institute of Bioscience and Biotechnology
The type strains of the species Paenibacillus ginsengisoli
(KCTC 13931T) and Paenibacillus anaericanus (DSM 15890T)
were compared in order to clarify the taxonomic relationship of
the two species. On the basis of 16S rRNA, gyrB and rpoB
gene sequence comparisons, the two strains shared 99.9, 100
and 99.4% similarity, respectively. The mean DNA-DNA
relatedness value was 71% and the genomic DNA G+C
contents were 42 and 41 mol%, respectively. Phenotypic data,
including fatty acid patterns and acid production, enzyme
activity and substrate utilization profiles, showed no
pronounced differences between the type strains of the two
species. These polyphasic data suggest that the two taxa
constitute a single species. According to Rules 38 and 42 of the
Bacteriological Code, they should be united under the name
Paenibacillus anaericanus, with the name Paenibacillus
ginsengisoli as a later heterotypic synonym.
[Supported by a grant from the KRIBB Research Initiative
Program]
A042
A043
Microvirga aerophila sp. nov. and Microvirga aerilata
sp. nov. Isolated from Air and Reclassification of
Balneimonas flocculans Takeda et al. 2004 as
Microvirga flocculans comb. nov., and Emended
Description of the Genus Microvirga
Hang-Yeon Weon1*, Soon-Wo Kwon1, Jung-A Son1,
Eun-Hye Jo1, Soo-Jin Kim1, Yi-Seul Kim1,
Byung-Yong Kim1, and Jong-Ok Ka2
1
Korean Agricultural Culture Collection, National Agrobiodiversity Center, National Academy of Agricultural Science,
Rural Development Administration, 2Department of Agricultural
Biotechnology, Seoul National University
Two bacterial strains 5420S-12T and 5420S-16T isolated from air samples,
were characterized using a polyphasic approach. 16S rRNA gene
sequence analysis showed that strain 5420S-12T was phylogenetically
related to Microvirga subterranea Fail4T (97.4% sequence similarity) and
Microvirga guangxiensis 25BT (97.1% similarity), and strain 5420S-16T
was closely related to Balneimonas flocculans TFBT (98.0% sequence
similarity) and Microvirga guangxiensis 25BT (97.2% similarity). The
G+C content of the genomic DNA was 62.2 mol% for strain 5420S-12T
and 61.5 mol% for strain 5420S-16T. The results of DNA-DNA
hybridization and phenotypic data showed that strains 5420S-12T and
5420S-16T could be distinguished from their phylogenetically related
species, and represent two novel species within the genus Microvirga, for
which the names Microvirga aerophila sp. nov. (type strain 5420S-12T =
KACC 11743T = NBRC 106136T) and Microvirga aerilata sp. nov. (type
strain 5420S-16T = KACC 11744T = NBRC 106137T) are proposed.
Furthermore, the reclassification of Balneimonas flocculans as
Microvirga flocculans comb. nov. is proposed and also emended
description of the genus Microvirga is provided.
A044
Bacillus composti sp. nov. and Bacillus suwonensis
sp. nov., Isolated from Cotton Composts
The First Report of Two Species of Polyporus
(Polyporaceae, Basidiomycota) from South Korea
Yi-Seul Kim1*, Soo-Jin Kim1, Rangasamy Anandham2,
Seung-Hee Yoo1, Hang-Yeon Weon1, and Soon-Wo Kwon1
1
Korean Agricultural Culture Collection, Agro-biodiversity
Center, National Academy of Agricultural Science, Rural
Development Administration, 2Department of Agricultural
Microbiology, Agricultural College and Research Institute, India
Eun Ju Woo1*, Jin Sung Lee1, Kyoung Hee Oh1,
Jae-Jin Kim2, and Young Woon Lim1
1
National Institute of Biological Resources, Environmental
Research Complex, 2Division of Environmental Science and
Ecological Engineering, College of Life Science and Biotechnology, Korea University
Two bacterial isolates from cotton composts in Korea,
designated strains 5M44T and 5M55T, were characterized using
a polyphasic approach. The cells were strictly aerobic, Grampositive, motile, spore-forming and rod-shaped. Phylogenetic
analysis of their 16S rRNA gene sequences revealed a clear
affiliation with the genus Bacillus. Strain 5M44T showed the
highest sequence similarities with Bacillus massiliensis
4400831T (97.3%) and Lysinibacillus xylanilyticus XDB9T
(97.2%), and the 16S rRNA gene sequence of 5M55T revealed
more than 97% with Bacillus massiliensis 4400831T (97.6%).
The G+C contents of the DNA of strains 5M44T and 5M55T
were 35.3 and 35.9 mol%, respectively. Major fatty acids
(>10%) of strain 5M44T were iso-C15:0 (38.0%), anteiso-C15:0
(28.4%) and iso-C16:0 (10.2%), and those of strain 5M55T were
iso-C15:0 (30.8%) and iso-C16:0 (18.1%). The major isoprenoid
quinone of both strains was menaquinone 7 (MK-7).
Comparative analysis of phenotypic and phylogenetic traits
indicated that strains 5M44T and 5M55T represented two novel
species of the genus Bacillus.
Based on morphological examination, two species of Polyporus, P. dictyopus and P. tuberaster, were identified, which
constitutes the first record of these species in South Korea. To
confirm their affinity within the genus Polyporus, the
phylogenetic relationships of Polyporus and allied genera were
established from nuclear large subunit ribosomal DNA (nLSU
rDNA) sequences, and a morphological diagnostic key is
presented to clarify the Korean species of Polyporus.
[Supported by the Korean indigenous species research project
from NIBR.]
[Supported by grants from RDA]
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A045
Characterization of Highly Unstable Phage SPG24
of Bacillus subtilis G24 and Its Long-Term Survival
Strategy in the Nature
Yu Ji Jang1*, Yong Sin Park2, Tae-Sook Kang3, and
Oh Hyoung Lee1
1
Department of Life Science, Mokpo National University,
2
Medical Laboratory, Mokpo City Medical Center, 3Department
of Biomedical Science Laboratory, Mokpo Science College
A phage SPG24 was isolated from deteriorating Chungkookjang using Bacillus subtilis G24 as host. B. subtilis G24 has
been isolated from grape to be used as soybean-fermenting
agent into Korean traditional food Chungkookjang. This phage
contains double-stranded DNA and seems to be a noble subtilis
phage because either a 400 base-pair SmaI fragment or a 850
base-pair EcoRV fragment of phage DNA was found to have
no match in Blast nucleotide sequence comparison. This phage
is belonging to group AI phages of B. subtilis according to its
morphology. This phage is highly virulent because it can cause
a rapid lysis of actively growing hosts after infection. Although
SPG24 is very unstable when it is exposed to outside of its host,
it can survive long in nature by hiding its chromosome in the
host spore and taking advantage of spore’s persistency. In spite
of its high virulency, this phage never causes a complete
demolition of its hosts in any growth volume. This would be
the reason why both virus and its host can survive together
until now evolutionally.
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Poster Sessions
B001
B003
Physiological and Metabolic Responses for Alkane
Degradation in Acinetobacter sp. Strain DR1
Genetic Diversity of Escherichia coli Obtained from
Yeongsan River
Jaejoon Jung* and Woojun Park
Division of Environmental Science and Ecological Engineering,
Korea University
Jeonghwan Jang1*, Tatsuya Unno1, Yaesul Suh1, and
Hor-Gil Hur1,2
1
Department of Environmental Science and Engineering,
Gwangju Institute of Science and Technology, 2International
Environmental Research Center, Gwangju Institute of Science
and Technology
The hexadecane degradation of Acinetobacter sp. strain DR1
was evaluated with changes in temperature and ionic salt
contents. Hexadecane degradation of strain DR1 was reduced
markedly by the presence of sodium chloride (but not
potassium chloride) and high temperature (37°C) was also
shown to inhibit the motility, biofilm formation, and hexadecane biodegradation. The biofilm formation of strain DR1
on the oil-water interface might prove to be a critical physicological feature for the degradation of hexadecane. The
positive relationship between biofilm formation and hexadecane
could be expected with changes in salts at 30°C, but not at low
temperatures (25°C). Alterations in cell hydrophobicity and
EPS production by temperature and salts were not correlated
with biofilm formation and hexadecane degradation. The
results of proteomic analyses have demonstrated that proteins
involved in fatty acid oxidation, the glyoxylate pathway, and
gluconeogenesis are highly upregulated and many oxidative
stress proteins have been identified as important for the
efficient biodegradation of hexadecane.
B002
Surface water samples were collected every month from 13
sites of Yeongsan river watershed from April to December
2009, and 60 E. coli isolates were obtained from each water
sample being doubted about fecal pollution. All E. coli isolates
were genotyped by Horizontal Fluorophore-Enhanced RepPCR DNA fingerprinting technique (HFERP) and discriminated at the strain level, then we surveyed the strain
composition of E. coli depending on each month and each site.
Overall, there was less diversity in isolates collected during
winter season compared to the other seasons. Average of
percentage of unique strain among isolate data sets on October,
November and December were below 30%, but over 50% from
April to September. Moreover the survey of genetic difference
between E. coli isolates obtained from surface waters and
sediments sampled on August and November 2009 was
conducted and interesting results were drawn, indicating that
there was no exchange between E. coli communities of surface
water and sediment on August, but E. coli of surface water
affected on the opposite side on November.
B004
Comparing Microarrays and Next Generation
Sequencing for Microbial Ecology Research
Food Waste Treatment by Using Thermophilic
Bacteria
Seong Woon Roh1*, Guy Abell2, Kyoung-Ho Kim1,
Young-Do Nam1, and Jin-Woo Bae1
1
Kyung Hee University, 2CSIRO
You-Jung Jung*, In-ho Chang, Ahnna Cho,
Dawoon Jung, and Tae-Seok Ahn
Department of Environmental Science, Kangwon National
University
Recent advances in molecular biology have resulted in the
application of DNA microarrays and next generation
sequencing (NGS) technology to the field of microbial ecology.
This review aims to: examine the strengths and weaknesses of
each of the methodologies, including depth of analysis,
throughput, ease of data analysis and cost effectiveness;
highlight the optimal application of each of the individual
technologies to the study of a particular environment; and
identify potential synergies between the two technologies,
whereby both sample number and coverage can be maximized.
We suggest that the efficient use of both microarray and NGS
technologies will allow researchers to advance the field of
microbial ecology and allow an improved understanding of the
role of microorganisms in their environment.
[Supported by grants from the Environmental Biotechnology
National Core Research Center, TDPAF, NMC0300837,
CAER, 21C Frontier Microbial Genomics and Application
Center Program, Eco-Technopia 21 project, KFDA and the
Office of the Chief Executive, CSIRO.]
For food waste treatment, we used thermophilic bacteria
isolated from humus leaf layer. Bacteria could be cultured at
45°C and their enzyme activities were characterized. And the
bacteria were identified based on their 16S rRNA gene
sequences. Then we applied those bacteria to food waste
treatment in pilot scale on various conditions. After than we
did it in real machine. Fourteen strains of isolated bacteria
were selected, based on their high enzyme activity. Those
bacteria were classified as family Bacillaceae, genus Bacillus
and genus Brevibacillus. In pilot scale, removal rate of food
waste was 46.1%/day. Optimal humidity was 75% for those
bacteria. Total bacterial number in treatment process was about
108 cells/g and pH about 4.5. In real machine, average of
removal rate was 96%/day in the best condition.
[This research was supported by Brain Korea21]
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B005
B007
Korean Human Gut Microbiota Revealed by
Barcoded Pyrosequencing
Flagella Motility is Involved in the High Current
Production of Geobacter sulfurreducens KN400
Young-Do Nam1,2*, Min-Soo Kim1,2, Kyoung-Ho Kim1, and
Jin-Woo Bae1
1
Department of Biology, Kyung Hee University, 2These authors
contributed equally to this work
Hana Yi*, Hoa Tran, Peng Zhou, Toshiyuki Ueki,
Kengo Inoue, Ashley E. Franks, and Derek R. Lovley
University of Massachusetts Amherst, USA
Human gut microbiota plays important roles in energy harvest
from diet, stimulation of intestinal epithelium, development
immune system, and regulation of fat storage of host.
Establishment of gut flora, however, has been limited to
western people and previously acquired information has not
been extensive enough to fully describe human gut microbiota.
In this study, we investigated gut microbiota of 21 Korean
using 454-pyrosequencing and compared with that of three
American, as well as temporal stability of intestinal microbiota.
Total 390,291 read sequences covering V1 to V3 hypervariable
regions of the 16S rDNA gene were analyzed. Overall shape of
microbial communities is dominated with previously mentioned
four phyla: Firmicutes, Bacteroidetes, Fusobacteria, and
Proteobacteria. PcoA (principal coordinate analysis) showed
that gut microbiota of Korean and American differed from
each other suggesting that genetic variation might affect the
composition of microbial community. Weighted and unweighted clustering analysis in Fast UniFrac revealed that the
composition of gut microbiota is maintained, but relative
abundance in microbial community is changed for 2 months
test period.
B006
In a recent study, a newly isolated Geobacter sulfurreducens
strain, designated as KN400, that dramatically increases power
output than earlier genome sequencing strain (DL1) was
reported. It was suggested that the enhanced current producing
capacity of strain KN400 could be originated from the changes
in outer surface components like as pili, flagella and exopolysaccharides. To evaluate the effect of motility system of strain
KN400 in its increased power production ability, knockout
mutants of flagella regulator, flagella hook protein and flagella
motor protein were constructed and characterized. The resulted
aflagellate and flagellated but nonmnotile mutants showed
remarkably slower and lower current production in microbial
fuel cell compared to wild type KN400. The pili and outersurface c-type cytochromes analysis also supported the
hypothesis that the defective electrode reducing ability of nonmotile mutants was not coming from pili or cytochrome
difference but from the absence of flagella motility.
B008
Change of Total Bacterial Number in Nutrient
Concentrating System Launched in Lake Soyang
Formation and Localization of Tellurium Nanocrystals in Shewanella spp.
In-Ho Chang1*, Eun-Young Seo1, You-Jung Jung1,
Seung-Ik Choi2, and Tae-Seok Ahn1
1
Department of Environmental Science, Kangwon National
University, 2Institute of Environmental Research, Kangwon
National University
Dong-Hun Kim1* and Hor-Gil Hur1,2
1
Department of Environmental Science and Engineering,
2
International Environmental Research Center, Gwangju Institute
of Science and Technology
Nutrients (nitrogen and phosphate) are necessary for plant
growth but are dissolved as low state in aquatic ecosystem. For
extracting nutrients from oligo-mesotrophic lakes, we invented
new devices with hydrophobic media, such as sponge and
rubberized coconut fiber (RCF). These devices were launched
at Lake Soyang on March, 2009, and then pore water of both
media and lake water were retrieved daily for 3 days and total
bacterial number and nutrient concentrations were measured.
Total bacterial numbers, nitrogen and phosphate concentration
in RCF were 4.3~8.4 × 106 cells/ml, 4.3 mg/l, 0.35 mg/l which
were about 11~26, 2, 4 and 2~3, 1.5, 2 times higher than those
of lake water (control) and sponge (comparative case),
respectively. These results suggest that RCF media have
excellent ability to accumulate the nutrient in the lake water
and bacteria are playing an important role for accumulating
nutrient in oligo-mesotrophic lake.
[Supported by grants from ECO-STAR project]
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Poster Sessions
Shewanella species are gram-negative facultative anaerobes
capable of utilizing a broad range of electron acceptors,
including several solid substrates such as Fe(III) and Mn(IV)
oxides. Shewanella species can reduce tellurite [TeO32-] to
elemental tellurium [Te(0)] under anaerobic conditions. This
reduction results in the formation of unique crystalline Te(0)
nanoarchitechtures as end products. The needle-like shaped
granules of element tellurium are localized in the cytoplasm or
near the cytoplasmic membrane, and each species forms a
similar structures. Several different S. oneidensis MR-1 deletion
mutants which are related with electron transfer cytochrome c
genes, produced tellurium nanostructures. However, they are
show different reduction rates. These results indicate that
deleted genes play a significant role and different mechanisms
are related in tellurite reduction.
[Support for this work from the 21C Frontier Microbial
Genomics and Applications Center Program (M102KK01001108K1101-01110), Ministry of Education, Science and Technology, Republic of Korea.]
B009
B011
Comparison of the Bacterial Community in
Drinking Water Purification Systems
Isolation of Plant Growth Promoting Bacteria from
Rhizosphere of Wild Plant at Barren Soil
Ok-Sun Kim*, Yunmin Kim, Kihyun Lee, Suk-Hwan Yoon,
Mincheol Kim, and Jongsik Chun
School of Biological Sciences, Seoul National University
Kyung-Mi Kim* and Hong-Gyu Song
Department of Biological Science, Kangwon National University
Despite the importance of its implication for human health, the
bacterial community composition in drinking water purification
systems has not well described. In the present study, we
compared bacterial communities in 20 different systems based
on 16S rRNA gene by traditional cultivation and barcoded
pyrosequencing. Sphingomonas spp., Staphylococcus spp.,
Mycobacterium spp., and Blastobacter spp. were isolated with
the sequence similarity of 98.1 to 100.0%. Although the
bacterial diversity in this habitat was relatively low compared
to natural ones, 19 known divisions and 18 unknown divisions
were detected, where Alphaproteobacteria with 54.8% were
overwhelmingly dominant. We also reveal the presence of
bacterial families with the members including environmental
relatives retrieved from diverse environments such as different
types of soil habitats, drinking water biofilm, freshwater
habitats and the atmosphere. Finally, we expect that complex
environmental factors could affect shaping the microbiological
composition of this environment. In conclusion, bacterial
community in the water purification system can be readily
monitored by pyrosequencing of 16S rRNA gene.
B010
This study was conducted with some rhizobacteria able to exert
beneficial effects upon plant growth. Many bacteria were
isolated from the rhizosphere of a wild plant Isachne globosa
(Thunb.) O. Kuntze, and among them two strains (AG and ED)
of plant growth promoting rhizobacteria (PGPR) were selected.
They could solubilize 531.5 mg l-1 of 0.5% insoluble phosphate
Ca3(PO4)2 in 3 days of incubation. Productions of indole-acetic
acid (IAA) by strain AG and ED under the addition of
tryptophan were 2.4 and 1.0 mg mg protein-1, respectively.
Strain AG and ED also produced other phytohormones, such
as gibberellin, abscisic acid (ABA) which were measured by
HPLC. They produced 270.1 and 271.7 mg l-1 of gibberellin,
respectively. Isolated bacteria were applied to the germination
test of wild plant in soil. Strain ED could enhance the
germination rate of seed of Abutilon avicennae by 26.6%.
Their plant growth promoting capabilities were examined in
the pot and microcosm composed of barren soil collected from
lakeside of Lake Paro, Kangwon-do. These results suggest that
the isolated strains of PGPR may be utilized as environmentally friendly biofertilizer for the revegetation of barren
lands.
B012
Isolation and Characterization of Glycerol-Utilizing
and Solventogenic Anaerobic Bacteria
Plant Growth Promotion by Isolated Strains of
Enterobacteria and Streptomyces
Jae-Hyung Ahn*, Chuloo Moon, Kyung-Duk Kim,
Byoung-In Sang, and Youngsoon Um
Clean Energy Center, Korea Institute of Science and Technology
Young-Mi Kim* and Hong-Gyu Song
Department of Biological Science, Kangwon National University
Glycerol is a byproduct of bioethanol and biodiesel production
processes and can be converted to more valuable products such
as ethanol, butanol, 1,3-propanediol, and 2,3-butanediol by
various anaerobic bacteria. In this study, we isolated glycerolutilizing anaerobic bacteria to obtain ones having a superior
ability to produce butanol. By adding acetic and butyric acids
into the mineral medium containing glycerol, we could enrich
butanol-producing bacteria from soil samples. Isolates showed
>99% 16S rRNA gene similarities with Clostridium diolis/
beijerinckii (group 1), C. butyricum (group 2), C. arbusti
(group 3), and Klebsiella oxytoca (group 4). The isolates
belonging to group 3 produced up to 9.3 g/L of butanol and the
isolates belonging to group 2 produced up to 17.1 g/L of 1,3propanediol from 30 g/L of glycerol. When glucose was used
instead of glycerol, the isolates belonging to group 1 produced
12.0 g/L of butanol and 6.9 g/L of ethanol and the isolate of
group 4 produced 21.0 g/L of 2,3-butanediol and 8.0 g/L of
ethanol from 60 g/L of glucose. This result shows the diversity
of glycerol-utilizing anaerobic bacteria and their potential for
industrial application.
Plant growth promoting bacteria have a number of mechanism
of growth enhancement such asvariousvarious phytohormone
production, phosphate solubilization, inhibition of plant
pathogen and others. We isolated enterobacteria Kluyvera
intermedia strain GBI and Ewingella americana strain GBL
and actinomycetes Streptomyces mirabilis strain GBQ from the
soil collected from Mt. Gyebang. These bacteria could produce
the plant growth hormones such as indole-3-acetic acid (IAA),
indole-3-butyric acid (IBA) and gibberellin. They also
inhibited growth of Fusarium oxysporum f. sp. lycopersici and
Fusarium oxysporum f. sp. niveum which are important
pathogenic fungi to many agricultural crops. E. americana
strain GBL showed the highest production rates of phytohormones, including 1105.1 µg mg protein-1 of IAA, 1313.9 µg
mg protein-1 of IBA and 799.3 mg l-1 of gibberellins (GA3).
The lengths of shoot and root of germinated tomato seedling
treated with strain GBL increased by 47.09 and 57.12%,
respectively in 21 days of incubation compared to the unionculated control. Dry weight of tomato plants grown was also
higher in the inoculated soil by 60.75% compared to that in the
uninoculated soil.
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B013
Plant Growth Promoting Capability of Isolated
Photosynthetic Bacteria
Hyeok-Do Kwon* and Hong-Gyu Song
Department of Biological Science, Kangwon National University
Environmental concern over conventional agricultural fertilezation and disease control measures has led to increased
interest in finding environmentally friendly alternatives. This
study was conducted with some photosynthetic bacteria for the
plant growth promotion. Some photosynthetic bacteria are able
to exert beneficial effects upon plant growth by production of
phytohormones. Phytohormones have essential roles in coordination of many growth and behavioral processes in the plant
life cycle. Several photosynthetic bacteria were isolated from
river sediments. Isolated photosynthetic bacteria, strain JFR
cultured in the modified Biebl and Pfenning medium, and
productions of indole acetic acid (IAA) and indole butyric acid
(IBA) were determined by colorimetric assay with Salkowski’s
reagent. Among the isolated bacteria, strain JFR showed the
highest production rate of 1388.1 µg mg protein-1 of IAA and
1251.4 µg mg protein-1 of IBA in the modified Biebl and
Pfenning medium. The effect of strain JFR on the germination
and growth of tomato seedlings were examined in a pot test.
This photosynthetic bacterium may be utilized as environmentfriendly biofertilizer in the agriculture.
B014
Plant Growth Promoting Effect of Isolated Rhizobacteria and Endophytic Bacteria
Sang-Yual Lee* and Hong-Gyu Song
Department of Biological Science, Kangwon National University
This study was conducted for the plant growth promotion by
some isolated rhizobacteria inhabiting the rhizospere of various
plants and endophytic bacteria residing within plant hosts
without causing disease symptoms. Rhizobacteria were
isolated from rhizosphere of mugwort, and endophytic bacteria
were isolated from the interior tissue of corn root using nutrient
broth medium. Indole-acetic acid (IAA) and indole-butyric
acid (IBA) production was determined by Salkowski’s reagent
test after cultivation of bacteria in nutrient broth containing
tryptophan, precursor of IAA and IBA. Rhizobacterial strain
LSY28 showed the highest production rates of 546.8 µg IAA
mg protein-1 and 609.2 µg IBA mg protein-1. Isolated endophytic bacteria were tested the antagonistic effects against
fungal pathogen. When the bacterial isolates were streaked
onto petri plate containing potato dextrose agar medium, strain
E23-2-2 showed the highest growth inhibition against
Fusarium oxysporum. This study demonstrated that bacterial
endophytes may also have beneficial effects on host plants
through biological control of plant pathogens.
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Poster Sessions
B015
Findings of Microbial Community Structure and
Dominant Species in Soils Polluted with Various
Contaminants
Jaisoo Kim1*, Sujeong Yang1, Hyunjung Kim2,
Jee-Young Kim2, and Sang-Seob Lee2
1
Department of Life Science, Kyonggi University,
2
Department of Biotechnology, Kyonggi University
This study is to examine what factors influence microbial
community structure by DGGE analysis and comparison using
soil samples collected from various contaminated sites. As a
result, the similarities based on DGGE band profiles showed
the closer relationship in regional properties than in pollution
characteristics, probably due to the degree of weak contamination. The highly contaminated samples with oil revealed
lower similarities with others in the same region or far
relationships with all the other samples in the other regions.
Thus the microbial community structure would more be
affected by region-based environmental factors than by
contamination factors in case of not serious pollution. All the
dominant culturable bacterial species were involved in
firmicutes or high GC Gram+ in a major portion of soil
samples and the highly oil-contaminated samples contained
Arthrobacter, Bacillus, Methylobacterium, Clavibacter, Streptomyces and Nocardia as reported genera, and Leifsonia as an
unreported genus.
B016
Biodegradation by Permeable Reactive Barrier
Using The Nano-Bio Hybrid Technology to Remediate
BTEX Contaminated Groundwater/Soil
Hye-Jung Lee*, Jaisoo Kim, Yoo-Taek Kim,
Seung-Gu Kang, and Sang-Seob Lee
Kyonggi University
Recently developed materials such as nano scale of zero valent
iron (NZVI) and emulsified NZVI droplet (ENZVID) have
been used in PRB process, but they can be inefficient process
that is not practical in many situations. It is believed that the
PRB using the nano-bio hybrid technique is the most economical and reliable method of response to this problem. The
technique is the fusion technology using the multi-nanopore
ceramic carriers and BTEX-degrading bacteria. To immobilize
cells, three kinds of carriers (compression molding, foaming
molding and circular carrier) were fabricated by pelletizing and
press molding methods. The compression molding carrier
showed the highest absorption rate among three carriers,
1.30×109, 1.15×108 and 5.05×108 cell/cm3 with P. putida BJ10,
E41, and their mixture after 5 days incubation at 28°C,
respectively. Immobilized BJ10 and E41 (mixture) could
degrade 76.4% of 40 mg/L BTEo-X for 4 days in a batch
system and 42.1% of 50 mg/L BTEo-X within 5 hours at 28°C
with 1.00×108 cell/cm3 (initial) in a continuous reactor system.
Furthermore we are going to experiment on reactor using the
waste-made ceramic carrier absorbed with BTEX- degrading
bacteria.
B017
B019
Heat Shock-Induced Plasmid Conformational
Changes in Rhodococcus sp. Strain DK17
Biogenic HgSe Nanoparticles Produced by Shewanella putrefaciens SP200
Kyungsun Kim1*, Sunme Shin1, Hae Youn Park1,
Dockyu Kim2, and Eungbin Kim1
1
Department of Biology, Yonsei University, 2Polar BioCenter,
Korea Polar Research Institute, Korea Ocean Research &
Development Institute
Cuong T. Ho1* and Hor-Gil Hur1,2
1
Department of Environmental Science and Engineering,
2
International Environmental Research Center, Gwangju Institute
of Science and Technology
Rhodococcus sp. DK17 possesses three linear megaplasmids
(380-kb pDK1, 330-kb pDK2, and 750-kb pDK3). The alkylbenzene-degrading genes are present on pDK2 while the
phthalate operons are duplicated and are present on both pDK2
and pDK3. DK 17 with a growth temperature optimum at 30°C
showed no growth at 37°C. When transferred to 30°C,
however, the 37°C culture began to grow immediately. This
indicates that the temperature of 37°C is not lethal but stressful
to DK17. Thus, to investigate the environmental factors affecting the structural stability of its megaplsmids, cells of DK17
were exposed to a heat shock of 37°C. Colonies were randomly
picked for colony PCR using primers specific for pDK2 and
pDK3, respectively. After screening 200 colonies a total of 30
strains were found to lose at least one of the three genes. PFGE
analysis revealed six different cases of plasmid conformational
changes: pDK2 loss (9), pDK3 loss (1), pDK2 loss with
appearance of a new plasmid (~700 kb)(10), pDK3 loss with
appearance of a new plasmid (~400 kb)(8), pDK3 loss with
appearance of a new plasmid (~600 kb)(1), and both pDK2 and
pDK3 loss with appearance of two new plasmids (~400 and
~700 kb)(1).
B018
Gram-negative facultative Shewanella are able to use metals
such as U(VI), Fe(III), As(V), Se(IV), Cr(III) as electron
acceptors, which have a great potential to apply in the environment technology for heavy metal treatment. Shewanella
putrefaciens SP200, the highest resistance strain to Hg (II), in
this report was used to investigate the ability of co-removing
Hg(II) and Se(IV) from aqueous environment, and the
production of HgSe nanoparticles as recovered nanostructure
products. This bacterium was incubated with 10 uM of Hg(II)
and 100 uM of Se(IV) at 30°C under aerobic condition in
defined medium amended with 10 mM of lactate. The SEM
and TEM images of samples collected after 5 days confirmed
spherical nanoparticles of Se(0) produced by Shewanella
putrefaciens SP200, and the EDX data showed the peak of
elemental Hg incorporated with Se nanospheres. Our results
suggest the possibility of recovering the toxic metals, Hg(II)
and Se(IV), as the advanced material by Shewanella
putrefaciens SP200.
[Support for this work from the 21C Frontier Microbial
Genomics and Applications Center Program, Ministry of
Education, Science and Technology, Republic of Korea.]
B020
Metagenomic Analysis of Uncultured Marine RNA
Viral Communities
Detection Method and Occurrence of Helicobacter
pylori in Water
Bandamaravuri Kishore Babu, Gui Hwan Han, Jang Min
Park, and Si Wouk Kim*
Pioneer Research Center for Controlling of Harmful Algal
Blooms (HAB) and Department of Environmental Engineering,
Chosun University
Eun-Sook Lee*, Gun-Seung Lee, Mok-Young Lee, and
Sun-Hee Han
Waterworks Research Institute, Seoul Metropolitan Government
Algal virus contributes a major role in controlling of algal
blooms in coastal environments. In the present study, metagenomic library has been constructed for an estimated average
RNA viral genome size of 10 kb at 5X coverage. Total genetic
material was extracted from the concentrated viral community
and was treated with DNase1 to remove DNA contamination.
The pure viral RNA was reverse transcribed using random
primers. The resulting cDNA was ligated with EcoR1 adapters
and amplified with adapter specific primers. The PCR product
of 600 bp-1200 bp size was cloned into TA vector and
transformed into E. coli. To check the insert, nearly about 20
primary clones were picked and sequenced. BLAST analysis
of the sequences obtained so far showed wide genetic diversity
and had no significant homology with the sequences in any
database. Further, we are continuing the effort to develop
molecular markers to study the distribution and abundance of
algal RNA viruses in costal aquaculture environments.
We studied the occurrence and method to detect H. pylori in
water using real-time PCR and selective cultivation. We
collected 103 samples containing surface water at the Han
River and wastewater from September 2009 to March 2010. H.
pylori were detected in two of 103 environmental water
samples using real-time PCR assay and their concentration was
5¯100 and 5.2¯101 cells. We used selective culture technique
to investigate the occurrence of live H. pylori for finished
water at the six water treatment plants and tap water at the
terminal sites of the distribution systems in Seoul. The
concentrated samples were spreading on columbia agar
containing horse blood and H. pylori-selective supplement and
were incubated at 37°C for 7 days under microaerophilic
atmosphere condition. Any H.pylori was not detected from 120
finished water and tap water samples.
[This study was supported by grants from the Waterworks
Research Institute, Seoul Metropolitan Government.]
[Supported by the Pioneer Research Program for Converging
Technology of the Ministry of Education, Science and
Technology, Republic of Korea]
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B021
B023
Isolation and Identification of Streptomyces sp.
AN090571 Producing Quorum Sensing Inhibitor
Distribution Patterns of the Members of Phylum
Acidobacteria
Jong-Ok Jang1,2*, Jin Hee Choi1, Jae-Chan Lee1,
Dong-Jin Park1, Chang-Keun Sung2, and Chang-Jin Kim1
1
Korea Research Institute of Bioscience and Biotechnology,
2
Department of Food Science and Technology Chungnam
National University
Sang-Hoon Lee* and Jae-Chang Cho
Department of Environmental Sciences, Hankuk University of
Foreign Studies
Quorum Sensing (QS) is community genetic regulation
mechanism that controls microbiological functions of medical,
agricultural and industrial importance. To screen novel quorum
sensing inhibitor (QSI) from actinomycestes, well diffusion
assay was used with Chromobacterium violaceum (CV026) as
a test organism and an isolate designated AN090571 was
selected. A comparative 16S rRNA gene sequence analysis
revealed that the strain AN090571 was belonged to the genus
Streptomyces and closest to Streptomyces misionensis NBRC
13063T with 99.01% of sequence similarity. AN090571 was
characterized morphologically and physiologically by the
given methods in the international Streptomyces project. It has
gray or yellow aerial hypha and showed high growth rate with
glucose and rhamnose. To investigate optimal carbon and
nitrogen sources for the high production of QSI, 20 carbohydrates and 14 nitrogen sources were tested separately and
carbon/nitrogen ratio was selected.
[Supported by technology Development Program for
Agriculture and Forestry, Ministry of Agriculture and Forestry,
Republic of Korea]
B022
Unexpectedly High Diversity of Bacterial
Community in Continental Shelf Sediment
Jin-Kyung Hong* and Jae-Chang Cho
Department of Environmental Sciences, Hankuk University of
Foreign Studies
We investigated the phylogenetic diversity of bacterial
community in deep marine sediment. Bacterial 16S rDNA was
amplified from community DNA extracted from continental
shelf sediment, and a 16S rDNA clone library was constructed.
The majority of the cloned sequences belonged to novel taxa,
and high diversity at phylum-level was observed. The most
dominant members of the clone library were phylogenetically
affiliated with Proteobacteria (35%) and minor groups
included Deferribacteres (3.8%), Gemmatimonadetes (1.9%),
Nitrospira (1.9%), and Planctomycetes (5.7%). These findings
demonstrate high diversity of bacterial community in the
continental shelf sediment, and expand our knowledge of
microbial diversity in deep marine sediment.
152
Poster Sessions
The distribution pattern of the phylum Acidobacteria, a
previously uncultured bacterial group, was investigated by
molecular ecological analysis. Acidobacterial 16S rDNAs were
observed in almost all soil samples, and members of acidobacterial primer group A were detected in all samples that
harbored the phylum Acidobacteria. Other primer groups, Y, G,
and O, showed limited distribution patterns. We further
divided the primer groups into acidobacterial subdivisions
(class-level). Subdivisional distribution patterns were determined by comparing the observed T-RFs with theoretical TRFs predicted by in silico digestion of acidobacterial 16S
rDNAs. Consistent with the PCR results obtained with
subgroup-specific primers, T-RFLP analyses showed that
acidobacterial subdivision 1 belonging to primer group A was
present in the majority of the soil samples. At the subdivisional
level, acidobacterial subdivision 1 might be the most widely
distributed group in this phylum, indicating that members of
subdivision 1 might be adapted to various soil environments,
and members belonging to other subdivisions might be
restricted to certain geographic regions or habitats.
B024
Simple and Rapid Detection of Listeria monocytogenes in Fruit Juice by Real-Time PCR without
Enrichment Culture
Hye-Jin Kim* and Jae-Chang Cho
Institute of Environmental Sciences and Department of
Environmental Sciences, Hankuk University of Foreign Studies
We developed a simple, rapid procedure for the detection of
Listeria monocytogenes in fruit juice using real-time PCR
without enrichment culture. PCR inhibitors were removed
from fruit juices using Chelex resin followed by gel filtration
with a Sephadex column. The purification step can be
completed in 20 minutes, and purified juice samples can be
directly used as PCR templates without further dilution. PCR
conditions were optimized and maximum sensitivity (ca. 1
cell/reaction) was achieved. This convenient method should
prove useful for high-throughput surveillance for L. monocytogenes as well as other food-borne pathogens that may
contaminate fruit juices.
B025
B027
Changes of Cultivable Bacterial Community of
Marine Sponges Depend on Media Composition
and Storing Period
Chatacteristics of Effective Dye Substance
Degrading Bacteria Obtained from the Complex
Dyeing Wastewater Treatment Plant
Hyun-Hee Cho* and Jin-Sook Park
Department of Biotechnology, Hannam University
Seon-Yeong Jin1*, Eun-Jung Heo1, Hong-Jae Choi1,
Eun-Joo Lee2, Young-Ok Lee1, and Gwang-Hui Lim2
1
Department Biological Science, College of Natural Science,
Daegu University, 2Department Environmental Engineering,
College of Engineering, Daegu University
The cultivable bacterial communities associated with three Jeju
Island Oedolgae marine sponges -Spirastrella abata, Spirastrella
panis, and an association of the sponges Poecillastra wondoensis
and Jaspis coreana in mixed cultures using F media with
inorganic composition and C media with organic compositionwere investigated by microbial community DGGE and 16S
rDNA phylogenetic analysis. Diverse bacterial group such as
α-, γ-, δ-proteobacteria, Bacteriodetes, Firmicutes were
cultured. δ-proteobacteria and Bacteriodetes were specific
bacteria cultivated in C media, but they were not cultivated in
F media which is inorganic media. The common bacterial
group was found to be α-proteobacteria, and the association of
two sponges showed the highest diversity among 3 sponge
samples. One month after marine sponges were collected and
freezed, the diversity of cultivable bacterial community
decreased. The common community was also changed to
Firmicutes. Therefore, the much difference of bacterial
diversity depending on media composition was not found. Also
we were able to confirm that the diversity of cultivable
bacterial community decreased depending on storing time of
sponge samples.
B026
Cultured Bacterial Diversity and the Impact of
Human Activity in Cryoconites Samples from Alps
Yung Mi Lee1*, So-Yeon Kim2, Jia Jung2, Eun Hye Kim1,
Kyeung Hee Cho1, Rosa Margesin3, Franz Schinner3,
Soon Gyu Hong1, and Hong Kum Lee1
1
Polar BioCenter, Korea Polar Research Institute, Korea Ocean
Research & Development Institute, 2Korean Minjok Leadership
Academy, 3Institute of Microbiology, University of Innsbruck,
Austria
The relationship between human impact and the diversity of
culturable bacteria in cryoconite samples from Alps was
investigated. Growth temperature, activity of protease and
lipase, and diversity were studied for 260 isolates. The ratio of
isolates which grew below 20°C was higher in samples with no
or weak human impact compared to that in samples with strong
human impact. In contrast, the ratio of isolates which grew as
high as 37°C were higher in strongly affected samples. In most
cases, protease activity at 10°C and 20°C seemed similar, but
the ratio of isolates with higher protease activity at 10°C than
20°C was higher when human impact were strong. In contrast,
correlation of lipase activity with human impact was not
evident. Isolates were included in the Actinobacteria, Bacteroidetes, Firmicutes, α-proteobacteria, ß-proteobacteria, and γproteobacteria and diversity was affected by human impact.
Isolates included in the Arthrobacter, Bacillus, and Pseudorhodobacter were detected only from samples with no human
impact while isolates included in the Enterobacteraceae,
Oxalobacteraceae, Pedobacter, Rhodococcus, and Sphingomonas were detected from samples with human impact.
In order to screen the complex dye degrading bacteria in the
wastewater of Daegu Dyeing Industrial Complex, bacteria were
isolated using dyeing wastewater itself as nutrient-medium
supplemented with mineral salts medium(MSM) and agar
(15g/L). The strong high pH of dyeing wastewater (pH 13) was
adjusted to pH7 for preparation of culture medium. After 3days
cultivation at 30°C, 2 strains grown on the dyeing wastewater
agar were pure cultured. For their identification 16S rRNA of
each strain was extracted and PCR was performed using both
341F and 518R primer. Those PCR products were sequenced
(~177 bp) and then compared with 16S rRNA sequences by
use of the EzTaxon. As a result, those bacteria was identified
as Pseudomonas monteilii (98.6%), P. taiwanensis (98.2%), P.
plecoglossicida (98.2%), P. mohhnii (98.1%), P. brenneri
(97.9%). Furthermore, optimal growth factors of those bacteria
in the complex dyeing wasterwater treatment plant such as
temp, pH and c-source will be discussed.
[This work (Grants No. 000367620109) was supported by
Business for Cooperative R&D between Industry, Academy,
and Research Institute funded Korea Small and Medium
Business Administration in 2009.]
B028
Phenotypic and Physiological Changes as a Result
of Natural Transformation in Acinetobacter oleivorans
DR1
Jungsoon Park* and Woojun Park
Division of Environmental Science and Ecological Engineering,
Korea University
The genus Acinetobacter is well-known to uptake exogenous
DNA from the environment. In this study, we provided
frequency of natural transformation of thirteen Acinetobacter
species along with novel oil- degrading Acinetobacter
oleivorans DR1 using a broad host range plasmids, pRK415
(tetA as selective marker). Many factors including cell-density,
DNA amounts, aeration are proven to be important for efficient
natural transformation. Interestingly, A. oleivorans DR1
(pRK415) displayed phenotypic and physiological differences
such as motility, ability for biofilm formation, response to
oxidative stress compared to wild type strain: transformed cells
became more sensitive to hydrogen peroxide and cumene
hydroperoxide and their motilities and biofilm formation
decreased. Oil biodegradation ability and growth fitness on
various substrates of transformed cells will be discussed. Our
data suggested that exogenous plasmid could influence on
either genes expression of host chromosome or membrane
integrity due to membrane-bound tetracycline resistant gene.
[This work was supported by a grant from NCRC (grant #:
20090091491).]
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B029
B031
Characterization of a Positive Transcriptional
Regulator of Isoeugenol Monooxygenase Gene from
Pseudomonas nitroreducens Jin1
1*
2
3
Ji-Young Ryu , Joong-Hoon Ahn , Michael J. Sadowsky ,
and Hor-Gil Hur1,4
1
Department of Environmental Science and Engineering,
Gwangju Institute of Science and Technology, 2Department of
Bioscience and Biotechnology, Konkuk University, 3Department
of Soil, Water, and Climate; and BioTechnology Institute,
University of Minnesota, USA, 4International Environmental
Research Center, Gwangju Institute of Science and Technology
We currently isolated about 140-kb DNA fragment containing
isoeugenol monooxygenase (iem) gene and its transcriptional
regulator gene, iemR gene from Pseudomonas nitroreducens
Jin1, which produced vanillin from isoeugenol. IemR played a
role as a positive transcriptional regulator for iem gene, which
encodes Iem involved in the biotransformation of isoeugenol to
vanillin in the presence of an inducer such as isoeugenol. In this
study, comparison of β-galactosidase activities of two E. coli
strains harboring pMC-IemR, which contains iemR and iem-lacZ
fusion gene, induced by isoeugenol showed that strain BL21
(DE3) exhibited about 2 times higher activity than strain DH5α.
In addition, β-galactosidase activity in BL21(DE3) was significantly induced even 0.1 µM of isoeugenol. Results from βgalactosidase assay of BL21(DE3)(pMC-IemR) incubated with
various aromatic compounds indicated that isoeugenol was the
best inducer for the expression of iem gene. β-Galactosidase assay
was also performed with sigma factor mutated E. coli strains
carrying pMC-IemR during culture. The sigma factor RpoH was
likely to be involved in the transcription of iemR or iem genes.
Effects of Ionizing Radiation and UV Irradiation on
Mutation Rate in Microorganisms
Ji-Hye Shin1*, Ji-hyun Nam1, Kyong-Hee Oh2,
Seungho Yu3, Myunju Lee3, and Dong-hun Lee1
1
Department of Microbiology, Chungbuk National University,
2
Department of Environmental Engineering, Chungbuk National
University, 3Korea Atomic Energy Research Institute
Advanced oxidation process (AOP) has shown substantial
potential as a substitute for the process treating wastewater
effluents containing toxic chemicals, xenobiotics, and etc.
AOPs well-known for their effectiveness include ozonation,
UV, γ-ray or electron beam (E-beam) irradiation. However,
these treatments can lead to serious secondary problems by
causing genetic changes in microorganisms. The goal of this
study was to evaluate the change in mutation rate of bacteria
exposed to ionizing radiation and UV. Mutation rates were
measured by Ames test and E. coli mutation test. DNA damage
was also estimated by umu-test. Death rate of bacteria exposed
to one of the ionizing radiations or UV was proportional to
irradiation dose. When bacteria were exposed with 5 mJ/cm2
UV, 0.5 kGy γ-ray and 0.2 kGy E-beam, the number of
mutants were 300, 150 and 60 times higher than that of
spontaneous mutation, respectively. The effects of E-beam on
both mutation rate and DNA damage were less than those of γray and UV irradiations. Therefore, application of ionizing
radiation, especially E-beam, appears to be more efficient for
wastewater treatment system.
[Supported by grants from MEST/ KOSEF]
B032
B030
Bacterial Communities in Oak-Sawdust Composting
Process for Lentinus edodes Cultivation
Seo-Yeon Yoon1*, Ji-Hyun Nam1, Chang-Duck Koo2, and
Dong-Hun Lee1
1
Department of Microbiology, Chungbuk National University,
2
Department of Forest Science, Chungbuk National University
Oak-sawdust composting process has been used as an
advanced method for Lentinus edodes cultivation. In spite of
its successful application to culture medium for L. edodes
cultivation, the bacterial population of leading the composting
process remains unknown. In this study, changes in composition of bacterial communities in the sawdust composting were
analyzed by PCR-T-RFLP and nucleotide sequences of 16S
rRNA gene. Temperature of sawdust piles has risen up to 60°C
during the composting process, and the numbers of total
bacteria and thermophilic bacteria in interior parts of sawdust
piles were higher than those in exterior parts. Structures of
bacterial communities were classified into three stages; the
sawdust composting stage, the pasteurization step, and the
mycelium growth phase. Predominant populations in the
composting process were members of Clostridium and
Amycolatopsis. Therefore, anaerobic fermentation appears to
be mediating oak-sawdust composting process.
[Supported by grants from KFS]
Fungal Community Analysis of the Samples Collected
from the Stone Chamber and Its Neighboring
Environment of the Takamatsuzuka Tumulus in
Nara, Japan
Kwang-Deuk An1*, Junko Tomita2, Tomohiko Kiyuna2,
Rika Kigawa3, Chie Sano3, Moriya Ohkuma1, and
Junta Sugiyama4
1
Japan Collection of Microorganisms, RIKEN BioResource Center,
Japan, 2TechnoSuruga Laboratory Co., Ltd., NCIMB group, Japan,
Independent Administrative Institution, National Research Institute
for Cultural Properties, Japan, 4TechnoSuruga Laboratory Co., Ltd.,
Chiba Branch Office & Lab, Japan
3
The fungal community analysis using the denaturing gradient gel
electrophoresis (DGGE) and clone library was applied to 50 and
22 samples were collected from the stone chamber interior, spaces
between the stone walls, and stone chamber exterior of the
Takamatsuzuka Tumulus (TT). The phylotaxa of Ascomycota,
Basidiomycota, Zygomycota, Chytridiomycota were detected from
these samples. Moreover, Eurotiales, Hypocreales, Chaetothyriales,
Helotiales, Saccharomycetales of the Ascomycota were detected in
a high ratio same as culture-dependent methods. Eurotiales and
Hypocreales were detected in a high ratio in each sample, but
Chaetothyriales was detected only interior a stone chamber and its
environment. The different phylotypes in Helotiales were detected
in neighboring environment of the stone chamber and burial
mound’s samples. Molecular phylogenetically, there were the
various ecological groups in samples derived from the TT stone
chamber and its neighboring environment.
[Supported in part by a Research Grant from the Institute for
Fermentation, Osaka (IFO), and Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology, Japan.]
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Poster Sessions
B033
Genomic Analysis of a Shigella sonnei
Bacteriophage
Kyoung-Ho Kim1*, Ho-Won Chang2, Young-Do Nam2,
Seong Woon Roh2, and Jin-Woo Bae2
1
Department of Microbiology, Pukyong National University,
2
Department of Life and Nanopharmaceutical Sciences and
Department of Biology, Kyung Hee University
Genomic structure of a bacteriophage that infects Shigella
sonnei was analyzed. The bacteriophage showed the morphology similar to the family Myoviridae, and the phylogenetic
analysis of sequences of g23 gene, major capsid gene showed
that it belonged to T4-like phage. The bacteriophage genome
was fully sequenced using 454 pyrosequencing and was ca.
170kb in length. Genomic comparison indicated that JS98, an
enterophage isolated from human stool was the closest relatives.
We compared two bacteriophage genomes and observed
several differences such as deletion, insertion and duplication.
Genomic sequencing helped us to understand the evolution and
relationship of bacteriophages.
[This work was supported by the 21C Frontier Microbial
Genomics and Application Center Program and the EcoTechnopia 21 project]
B034
Roles of Effective Microorganisms in Bioremediation
of the Contaminated Harbor Sediments
K. I. Ekpeghere1*, B.-H. Kim2, and S.-C. Koh3
Department of Civil and Environmental Engineering, Korea
Maritime University, 2Environmental Biotechnology Research
Center, Korea Research Institute of Bioscience and Biotechnology, 3Department of Environmental Engineering, Korea
Maritime University
1
B035
Enrichment and Competition of Methanogens and
Sulfate-Reducing Bacteria in Sterilized Tidal Flat
Under Diverse Incubation Conditions
Kihyun Lee*, Ok-Sun Kim, and Jongsik Chun
School of biological sciences and Institute of Microbiology,
Seoul National University
Tidal flat is known to contain dissovlved methane upto
hundreds nM concentration. Microorganisms mediating
methane fluxes have been intensively studied in deep-sea
sediments, but rarely in tidal flats. We aimed to cultivate
naturally abundant methanogens in tidal flats, and observed
competition between methanogens and sulfate reducers under
diverse incubation conditions. The hypothesis was that
physical structure of sterilized bulk sediment could generate
various realizable niche in the bottle and prevent radical
simplification of community structure, so that we can enrich
naturally abundant microorganism by means of dilution.
Bacterial and archaeal 16S rRNA, mcrA and dsrA genes were
quantified in each natural and incubated sample. In natural
samples, bacterial 16S rRNA gene was 7-20 times more
abundant than archaeal 16S rRNA gene and correlated with
dsrA abundance, while archaeal 16S rRNA gene did not with
mcrA. After incubation for over a month, archaeal dominance
was observed in hydrogen-enriched incubations, not exactly
correlated by mcrA abundance. In various conditions, dsrA and
mcrA abundance showed a roughly complementary pattern
except in a methane-enriched incubation.
B036
Idiomarina taeanensis sp. nov., a Novel Marine
Bacterium Isolated from Crude Oil-Contaminated
Seawater
Young-Hyun Park1*, Dong-Heon Lee1,2, Young Sun Lee1,
Jae Sung Jung1, and Hyung-Yeel Kahng1
1
Sunchon National Univeristy, 2Pohang University of Science
and Technology
We applied Effective Microorganisms (EM) carried on loess to
the treatment of sediments contaminated with organics. We
measured the physiochemical, biochemical and microbial
parameters during the remediation process at a lab scale.
Treatment with higher concentrations of EM loess balls (4%)
and with a lower concentration of EM (0.1%) containing
molasses (0.05%) contributed to a more rapid removal of
malodor. Acetic acid, propionic acid, valeric acid, caponic acid,
and lactic acid were rapidly removed when molasses (0.05%,
w/w) were used as a supplemental nutrient source, indicating
the EM activity was enhanced by molasses. Fermentation of
molasses by EM showed that acetic acid was produced much
higher than the other organic acids and most of the organic
acids appeared to be eventually converted through intermediate
metabolites to acetate. This shows that the rapid reduction in
malodor could be due to the reduction of these organic acids by
the molasses-assisted EM. It was concluded that EM might be
directly involed in biodegradation of orgninc materials in the
sediments.
A novel, strictly aerobic, motile, Gram-staining-negative
bacterium, designated strain MWD-44T, was isolated from the
coastal seawater of Taean in Chungnam, Korea. It grew at 1042°C, at pH 5.0-10.0 and in the presence of 1-12% NaCl. A
comparative 16S rRNA gene sequences study revealed that
strain MWD-44T belonged to the genus idiomarina and shared
significant similarity with Idiomarina aestuarii (99.5%) and
Idiomarina salinarum (97.1%), respectively. DNA-DNA
relatedness values between strain MWD-44T and the type
strains of Idiomarina aestuarii KYW314T and Idiomarina
salinarum CCUG 54359T were 44.5 and 31.8%, respectively.
The predominant cellular fatty acids were iso-C15:0 (22.6%),
Summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c, 13.7%),
and C16:0 (10.4%). The G+C content of the genomic DNA was
47.9 mol% and the major respiratory quinone was ubiquinone
8 (Q-8). Based on phenotypic and genotypic data, strain
MWD-44T represents a novel species within the genus
Idiomarina, for which the name Idiomarina taeanensis sp. nov.
is proposed. The type strain is MWD-44T (= KCTC 23070T =
JCM 16452T)
[Supported by grant from Center for Yeongdo Coastal Research,
Korea Maritime University]
[Supported by the Ministry of Environment (Grant No. 200805001-0033-0)].
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B037
B039
Biodegradation of Oils by Pseudomonas taeanensis
MS-3
Isolation and Characterization of Lactococcus lactis
HK-9 Derived from Feces of a New Born Infant
Dong-Heon Lee1,2*, Kye-Heon Oh3, and
Hyung-Yeel Kahng1
1
Sunchon National University, 2Pohang University of Science
and Technology, 3Soonchunhyang University
Hyun Baek*, Hye-Ran Ahn, Yun-Jin Song, Yun-Seok Cho,
and Kye-Heon Oh
Department of Biotechnology, Soonchunhyang University
Pseudomonas taeanensis MS-3T isolated from oil-contaminated seashore site has been reported as a novel species to
degrade oils, which shared sequence similarity to Pseudomonas
marincola KMM 3042T (97.9%), Pseudomonas cuatrocienegasensis 1NT (97.8%), Pseudomonas borbori LMG 23199T
(97.3%), and Pseudomonas lundensis KACC 10832T (97.1%)
as the closest neighbors (in press, Int. J. Sys. Env. Microbiol.,
2010). GC-MS analysis revealed that approximately 80% of
0.5% gasoline was removed by strain MS-3T with 7-days
incubation at 20°C. Strain MS-3T was also able to completely
degrade 0.5% diesel and 0.5% kerosene at the same incubation
time point. Analysis of substrate-dependent growth pattern
revealed that strain MS-3T grew to an optical density of 0.25 at
600nm without lag phase with kerosene as a sole carbon source,
while in BM medium containing 0.5% gasoline or 0.5% diesel
as a sole carbon source it grew after approximately 2-days lag
phase. PAH ring-hydroxylating dioxygenase and alkane
hydroxylase responsible for the biodegradation of oils in strain
MS-3T were also cloned and characterized
The purpose of this work was to characterize the bacterium,
Lactococcus lactis HK-9 isolated from the feces of a 2-day
newborn infant. Initially, the physiological and biochemical
characteristics of the strain HK-9 was examined. Both
BIOLOG system and phylogenetic analysis using 16S rRNA
sequencing were performed to identify and designated as L.
lactis HK-9, and registered in GenBank as [GU936712].
Phylogenetic analysis of L. lactis HK-9 was plotted based on
16S rRNA sequence comparisons. The changes of bacterial
growth, production of lactic acid and acetic acid as metabolites,
and pH were monitored. Maximum concentrations of lactic
acid and acetic acid reached 495.6 mM and 104.3 mM,
respectively, and the initial pH 7.0 of the cultures decreased to
4.1 after incubation of 60 hrs. Significant antimicrobial activity
of the concentrated supernatants against 4 Gram-positive and 5
Gram-negative bacteria was demonstrated by the plate
diffusion method. The results of this work are applicable to the
elucidation of defense mechanism of various pathogens in
newborn infant.
[Supported by Ministry of Environment, Grant No. 200805001-0033-0].
B038
B040
Antibacterial Effects and Cellular Responses of
Imipenem-Resistant Pseudomonas aeruginosa
Exposed to Green Tea Polyphenols
*
Yu-Jin Song , Su-Hee Cho, Yun-Seok Cho, and
Kye-Heon Oh
Department of Biotechnology, Soonchunhyang University
The aim of this work was to investigate the bactericidal effects
and cellular responses of green tea polyphenol (gTPP) and
imipenem on Pseudomonas aeruginosa. Imipenem-resistant Ps.
aeruginosa was isolated from patient in hospital. The
bactericidal effects of gTPP and imipenem were evaluated on
the basis of its minimum inhibitory concentrations (MIC). The
combined use of gTPP and imipenem resulted in ~16-fold
reductions in the MICs of imipenem for the imipenemsusceptible/resistant Ps. aeruginosa, respectively. The bactericidal effects of the imipenem and gTPP was evaluated using
the time-kill assay. Synergetic effects of the combinations of
gTPP and imipenem against Ps. aeruginosa were confirmed.
Western blot was performed to investigate the expression of
stress shock proteins in the strains exposed to gTPP. SDSPAGE revealed that the amount of lipopolysaccharides increased
or decreased in the strain treated to different concentrations
and exposing periods of gTPP. SEM demontrated the presence
of umbilicated and wrinkled surfaces for cells treated with
gTPP or imipenem.
156
Poster Sessions
The Effect of Methane on The Microbial Community
in The Sediment of Ulleung-Basin
Jin-Woo Lee1*, Kae Kyoung Kwon1, Jang-Jun Bahk2, and
Jung-Hyun Lee1
1
Marine Biotechnology Research Centre, Korea Ocean Research
& Development Institute, 2Korea Institute of Geoscience and
Mineral Resources
Geophysical survey has shown that gas hydrate exists in deepsea sediments in the East Sea, Korea. Bacterial community
analysis in methane hydrate-bearing deep-sea sediments (ca.
8m below the seafloor) obtained from the Ulleung-Basin was
made by T-RFLP and 16S rDNA pyrosequencing. In the TRFLP analysis, it was shown that the community structure in
the 'possible' sulfate methane transition zone (SMTZ) that was
characterized as depleted sulfate and increased methane, was
different from that of the upper and deeper region of the SMTZ.
And then, pyrosequencing analsys has been conducted to show
the bacterial community structures in high-resolution. Among
the total sequences, about 62% reads were sorted and analysed.
The homology searches revealed that the most of those
sequences, more than 50% affiliated into the Chloroflexi and
candidate division JS1, which were characteristic in the deepsea marine sediments containing methane hydrate and that the
proportion of sequences within δ-proteobacteria was relatively
higher in the SMTZ (100∼150cm) and surface region
(0∼20cm). This result implied that microbial community
structure in the SMTZ characteristic in microbial community
structure.
B041
B043
Biodiversity and Phylogenetic Analysis of Antibiotic
Streptomyces Isolated from Various Environments
Hyo-Jin Lee1* and Kyung-Sook Whang1,2
Department of Microbial & Nano Materials, Mokwon University,
2
Institute of Microbial Ecology & Resource, Mokwon University
1
We have hitherto isolated large numbers of antifungal and
antibacterial actinobacteria from various environments,
including different layers of bamboo forest soils, livestock
composts and heavy metal polluted soils. We enumerated the
actinobacterial densities, the number of actinobacteria in litter
layers of bamboo and some of composts were two to three
orders of magnitude greater than those enumerated in other
environmental samples. In the course of screening of antibiotic
activity against seven plant pathogenic fungi and two plant
pathogenic bacteria of 530 isolates, 110 strains showed
antibiotic activity. Ninety-five percent of the total antibiotic
isolates were Streptomyces by 16S rRNA gene sequence
analysis. The 105 Streptomyces, categorized into four clusters
based on the Benjamin’s classifying (2005). The Streptomyces
from bamboo forest soils were belonging to the cluster II
(72%), I (6%), III (8%), IV (14%). On the other hand, 86% of
total Streptomyces from livestock composts was cluster I.
Phylogenetic analysis indicated that bamboo forest soil
valuable in detecting diverse antibiotic Streptomyces in the
various environments.
Comparative Analysis of Microbial Community
Depending on the Cell Size from the Bathypelagic
Layer of Ulleung-Basin, Korea
Mi-Ree Kim*, Kae Kyoung Kwon, and Sang-Jin Kim
Marine Biotechnology Research Center, Korea Ocean Research
& Development Institute
The size of microorganisms varies considerably. At the
smallest end of this size spectrum are the ultramicrobacteria(UMB) which has a cell volume of less than 0.1μm3,
irrespective of growth conditions. UMB are highly abundant in
marine environments and they play important roles in the
global nutrient cycle and in the marine food chains. In the
present study the microbial diversity in the bathypelagic layer
of Ulleung-basin was compared by the size fraction which is
lager than 0.2μm(non-UMB) or smaller than 0.2μm(UMB). A
total 420 clones of archaeal group and 303 clones of bacterial
group were analyzed by 16S rRNA gene target sequence. All
archaeal clones belong to Crenarchaeota and Euryarchaeota
and a majority of both fractions was affiliated with
Crenarchaeota marine group I by 82-85%. Bacterial clones
were derived from 5 major lineages, α-,β-,γ- and δ-Proteobacteria and Chloroflexales. γ-Proteobacteria represents a
dominant bacterial group in the fraction of non-UMB and
UMB by 85% and 78%, respectively. Most clone of UMB is
grouped into the uncultured group and these results support
UMB possess high frequency of unique microbial community
compared to non-UMB.
[Supported by MEGRC]
B042
B044
Phylogenetic Characteristics of Exopolysaccharide
Producing Bacteria from Root System of Angelica
sinensis
1*
1
1,2
Hae Ran Lee , Song Ih Han , and Kyung Sook Whang
1
Department of Microbial & Nano Materials, Mokwon University,
2
Institute of Microbial Ecology & Resources, Mokwon University
In the previous study, we have been detected exopolysaccharide(EPS) producing bacteria in rhizosphere soils of
domestic medicinal herbs. Half of the total isolates from
rhizosphere soil of Angelica sinensis was EPS producing
bacteria, suggesting the dominance of EPS producing bacteria
in rhizosphere soil of Angelica sinensis. In the present work, 35
EPS strains were isolated from root system (rhizosphere,
rhizoplane, endophyte) of Angelica sinensis. The 16 EPS
strains from rhizospere soil belonging to the genus Burkholderia, Paenibacillus, Mucilaginibacter, Rhodococcus, Variovorax, Achromobacter, Shinella, Rhizobium, and Mesorhizobium. Forty-two percent of the total EPS strains from
rhizoplane was dominantly distributed Rhizobium-related
genera. Three EPS strains were genus Chitinophaga from
inside of root. In particular, DR14 and DRP35 produced high
levels of exopolysaccharide in 4% of glucose (6,555mpa.s) and
2% of sucrose (3,275mpa.s), respectively. When determine by
homology relationship of the 16S rRNA gene sequence with
the relative taxa, the DR14 strain was closely Burkholdria
caribensis (99%), and DRP35 strain was closely Terriglobus
sp. (96.8%).
Proteome Analysis of Halophilic Bacterium, US6-1
Isolated from PHAs Contaminated Environment
Sung Ho Yun1*, Chi Won Choi1, Sang Oh Kwon1,2,
Dong Gyu Lee1, Kae Kyoung Kwon3, Young Ho Chung1,
and Seung Il Kim1
1
Division of Life Science, Korea Basic Science Institute,
2
Graduate School of Analytical Science and Technology,
Chungnam National University, 3Microbiology Laboratory,
Korea Ocean Research & Development Institute
Contamination of marine sediments with PHAs (polycyclic
aromatic hydrocarbons) is critical environmental problem
because PHAs have toxic, carcinogenic, and genotoxic properties. Therefore, bioremediation of PAHs from contaminated
environments is very important. Halophilic bacterial strain
US6-1 is a gram-negative bacterium and has high sequence
homology with Novosphingobium. This strain US6-1 can
utilize several polycyclic aromatic hydrocarbons(PAHs) such
as pyrene, benzo[a]pyrene, phenanthrene, et al. Recently,
genome sequencing of strain US6-1 was finished and the draft
data is available. Purpose of this project is to search of
enzymes of PHAs biodegradation pathways in stain US6-1. In
this study, proteomes of stain US6-1 cultured under several
PAHs as carbon sources were analyzed by 2DE and MS/MS
analysis. We detected about 500 protein spots on the 2-D gel in
control medium(MM2) and found several differentially
induced protein spots in different PAHs conditions. Identification of these proteins and their biological significance were
presented. We suggest that these proteins are related to the
PHAs biodegradation pathway and physiological characterization of stain US6-1.
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B045
B047
Metagenomic Analysis of Microbiomes of a Korean
Traditional Fermented Kimchi
Bacterial Community Analysis of Asian Dust:
Cultivation-Dependent Approach
Ji Young Jung1*, Hyo Jung Lee1, Jin-Woo Bae2,
Yoonsoo Hahn1, and Che Ok Jeon1
1
Department of Life Science, Chung-Ang University,
2
Department of Biology, Kyung Hee University
Hang-Yeon Weon1*, Jung-A Son1, Wan-Gyu Kim2,
Soon-Wo Kwon1, Soo-Jin Kim1, Yi-Seul Kim1, and Jong-Ok Ka3
1
Korean Agricultural Culture Collection, National Agrobiodiversity Center, National Academy of Agricultural Science,
Rural Development Administration, 2Agricultural Microbiology
Division, National Academy of Agricultural Science, Rural
Development Administration, 3Department of Agricultural
Biotechnology, Seoul National University
Kimchi is well known as a traditional and popular Korean
fermented food. In this study, we monitored biological states
during a kimchi fermentation process using a metagenomic
analysis and we mainly focused on microbial community
change and their metabolism. For metagnomic analysis, the
kimchi soups containing microorganisms were periodically
collected for genomic DNA extraction. The extracted DNA
samples were analyzed using the 454-FLX technology, which
generated total 701,556 sequences with an average read length
of 433.4 bp. Community analysis based on reads containing
16S rRNA genes showed that the Kimchi microbiome was
predominated by the genera Leuconostoc, Lactobacillus, and
Weissella and the microbial community was changed
dramatically during the fermentation periods. We analyzed
COGs of the metagenomes, which showed that their
metabolisms corresponding to COG categories were also
changed during the fermentation. And comparison of random
metageome reads with previously sequenced several LAB
genomes showed high mapping coverage.
[This work was supported by Technology Development Program for Agriculture and Forestry and EB-NCRC program.]
B046
Much attention has been given to the nature of Asian dust and
its effect on the Korean Peninsula in the recent years due to an
increase in the intensity and frequency of Asian dust events.
However, there has been little research on the distribution
characteristics of airborne bacteria in Asian dust. To address
this issue, the quantities and compositions of airborne bacteria
were monitored by using a culture-dependent technique. The
bacterial concentrations of the normal atmosphere (NA) varied
with the sampling dates and locations, and were in the range 41,148 CFU/m3 during the study period. However, when an
Asian dust event occurred, the bacterial concentrations
increased sharply at a rate proportional to the intensity of the
Asian dust event. A rarefaction curve and diversity index
analysis showed that the bacterial diversity of the normal
atmosphere was higher than that of the dust samples from
Korea and China. In a community tree, the Asian dust samples
were clearly separated from NA and source-region soil
samples. Consequently, the airborne microorganisms detected
in Asian dust events were quantitatively and qualitatively
different from those in the normal atmosphere.
B048
Microbial Diversity of Sediments at Gas Seep Area
of Kagoshima Bay as Determined by Pyrosequencing
Bacterial Community Analysis Using Pyrosequencing in
Chonggak Kimchi during Fermentation
Hyun Hee Cho1*, Kae Kyoung Kwon1, Chiaki Kato2,
Takako Sato2, and Sang-Jin Kim1
1
Korea Ocean Research & Development Institute, 2Institute of
Biogeosciences, Japan Agency for Marine-Earth Science and
Technology
Hyo Jung Lee1*, Ji Young Jung1, Eun Jin Park2,
Jin-Woo Bae2, and Che Ok Jeon1
1
Department of Life Science, Chung-Ang University,
2
Department of Biology, Kyung Hee University
Bacterial community structure was compared between two
different sediments based on the 16S rRNA gene sequence
analysis by pyrosequencing method. The sediment samples
were collected from tube worm inhabited area (MEGC0044)
and caldera region (MEGC0046), which are located at shallow
water of marine hydrothermal vent area in Kagoshima Bay,
Japan. The phylotypes, the class δ-, ε- and γ-Proteobacteria,
and Flavobacteria are predominant in both sediments but the
proportion was different. Other phylotypes, Acidobacteria,
Chloroflexi, Planctomycetes, and the candidate division WS3,
also could be detected by more than 1%. The predominance of
δ- and ε-proteobacteria represents distinct characteristics
compared with the bacterial community structure previously
reported from other cold seeps or hydrothermal vent areas
where the γ-Proteobacteria is dominant. The proportion of εproteobacteria in MEGC0044 (28.6%) was much higher than
that of δ-proteobacteria (14.8%) but vice versa in MEGC0046
by 13.5 and 20.6%, respectively. The results imply that the
sulfur oxidizing is major biogeochemical process in
MEGC0044, however, MEGC0046 is dominant by the sulfur
reducing process.
[Supported by MEGRC]
158
Poster Sessions
Chonggak kimchi is a Korean traditional fermented food using
radish. In order to analyze bacterial communities of Chonggak
Kimchi,kimchi soup samples containing microorganisms were
collected every three days for one month via through of
sterilized gauzes. The pH decreased from 5.8 to 4.5 during 21
day-fermentation, and was then maintained constantly around
pH 4.5 ± 0.05. The multiplex barcoded pyrosequencing was
performed in a single run, with multiple samples tagged
uniquely by multiplex identifiers, using universal primers 27F
and 517R. Phylotypes were identified by using naïve Bayesian
rRNA classifier at the Ribosomal Database Project website
(http://rdp.cme.msu.edu/classifier/classifier.jsp). The results
showed that various bacterial groups such as Lactobacillus,
Streptophyta, Leuconostoc, Bacillus, Sphingomonas, Pseudomonas, and Serratia were existed in Chonggak Kimchi at 0
day. On the process of fermentation, bacterial community
became simple; the most predominant bacterial group was the
genus Leuconostoc, and the second was Lactobacillus.
[This work was supported by Technology Development
Program for Agriculture and Forestry and EB-NCRC.]
B049
Bacterial Community Analysis of Asian Dust:
Cultivation-Independent Approach
Hang-Yeon Weon1*, Jung-A Son1, Chan-Won Park2,
Jae-Ho Joa3, Hyung-Jun Noh4, Chang-Muk Lee5,
Soon-Wo Kwon1, Jong-Ok Ka6, Soo-Jin Kim1, and Yi-Seul Kim1
1
Korean Agricultural Culture Collection, National Agrobiodiversity
Center, National Academy of Agricultural Science, Rural Development
Administration, 2Soil & Fertilizer Management Division, National
Academy of Agricultural Science, Rural Development Administration,
3
Agricultural Research Center for Climate Change, National Institute of
Horticultural & Herbal Science, Rural Development Administration,
4
Mushroom Research Division, National Institute of Horticultural &
Herbal Science, Rural Development Administration, 5Functional BioMaterial Division, National Academy of Agricultural Science, Rural
Development Administration, 6Department of Agricultural Biotechnology,
Seoul National University
To elucidate the bacterial diversity and their relative abundance in
Asian dust samples collected in Korea and China, we used
massively parallel pyrosequencing The diversity and composition
of the dust-borne bacterial communities were relatively static at the
phylum level. These dominant phyla were represented in the source
region soils, reflecting that dust bacteria might have mainly
originated from the source regions soils, but the composition was
significantly different. OTU-based analyses such as rarefaction,
diversity indices, and Venn diagrams showed that many differences
were observed across sampling locations and year of Asian dust. In
addition, community trees showed that differences between the
sampling locations were more pronounced than differences between
years. Hence, our results indicated that Asian dust might harbor a
unique community that possibly originates from the source region
soils and is shaped by selective forces during long-range transport
and may vary depending on sampling sites and dates.
B050
B051
Efficient Bioremediation of High Concentrated
Tetrachloroethylene by Mixed Cultures of Bacteria
in Anaerobic Condition
Jin Ha Lee*, Jaisoo Kim, and Sang-Seob Lee
Department of Biological Engineering, Kyonggi University in
Korea
Tetrachloroethylene (PCE) is widely used in many industrial
fields, usually as a mixture with other chlorocarbons, which
can cause central nervous system depression. Bioremediation
is one of the efficient process to overcome this pollutant
evidently. We isolated a few bacterial mixtures which showed
degradation of PCE in anaerobic condition. Among those, we
selected high efficient mixtures named as Pe1, Pm1, Pm2, Pm3
and Po3. Ethanol and methanol were used as a carbon source
for the growth of the bacteria. The efficiency to degrade high
concentrated (50 mg/L of initial concentration in minimal
medium with yeast extract(1g/L), cell concentration 1.0 g/L,
pH 7 and temperature 30°C) PCE under strict anaerobic
condition was 75.8~ 80.1% in 30 days. We also found some
fermentable bacterial mixtures which could produce hydrogen
gas. The Po3 and fermentable mixtures showed more efficient
removability of PCE. Furthermore we also screened the
capability with mixed bacterial culture in aerobic condition,
some mixtures turned PCE to trichloroethylene (TCE) with
methanol, phenol and toluene as a carbon source, which has
not been reported earlier.
B052
High Throughput Cultivation of Major Marine
Bacterial Lineages Using Various Culture Conditions
from the East Sea
A Metagenomic Screen Identifies Genetic Elements
that Encode Novel Determinants for Bacterial
Biofilm Formation
Seung-Jo Yang*, Kwang-Hyun Rhee, Hyun-Myung Oh, and
Jang-cheon Cho
Division of Biology and Ocean Sciences, Inha University
Mi Young Yoon* and Sang Sun Yoon
Class of Microbiology, College of Medicine, Yonsei University
High-throughput cultivation (HTC) based on dilution-toextinction has been successfully used to cultivate marine
uncultured bacterial lineages from Oregon coast and the
Sargasso Sea. In this study, we applied a modified version of
HTC to cultivate major marine bacterial lineages in the East
Sea for one year. Microbial growth was detected in 1446 wells
among 7872 inoculated wells and 325 colony-forming isolates
were obtained on agar plates by transferring extinction cultures.
Based on phylogenetic analyses of 16S rRNA gene sequences
from pure isolates, cultured strains were generally assigned to
the SAR11 groups 1 and 3, Roseobacter clade and SAR92
group. The isolates belonging to the OM42, OM60, OM182,
K189A clades and the phylum Lentisphaerea were also
recovered. Interestingly many strains were affiliated to genera
of Fucophilus, Coraliomargarita and Pelagicoccus in the
phylum Verrucomicrobia. This study suggests that efficient
cultivation of novel members of major marine bacterial groups
could be facilitated by a long-term approach using various
culture conditions mimicking natural environments.
[Supported by the 21C Frontier Program of Microbial
Genomics and Applications]
Metagenomics is a new research field, in which a collection of
genetic materials recovered from a certain microbial ecosystem
is studied.Bacteria propagate as complex, highly organized
communities known as biofilm. We sought to identify novel
genetic elements, presence of which resulted in the enhanced
biofilm formation. To this end, we screened a library that
consists of 5,760 E. coli DH10B clones, each of which harbors
a bacterial artificial chromosome prepared from DNA
extracted from the large-bowel microbiota of BALB/c mice.
We successfully identified 7 clones. Interestingly, the insert
DNA from three clones possibly originated from uncultivated
bacteria, but the gene from the remaining clone resembled
sequences reported in Bacteroides sp. and Filobasidiella sp.,
with low similarity. Our results represent a successful application
of metagenomic approach to reveal a novel mechanism for
bacterial biofilm formation.
[This study was supported by Yonsei University College of
Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Reserach Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology, 7-2009-0524 to SSY.]
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B053
B055
Diversity of Fungal Endophytes in Leaves and Stems
of Red Pepper (Capsicum annuum L.) Cultivated in
Greenhouses and Open Fields
Fusarium spp. Isolated from Wilted Gypsophila
(Gypsophila elegans B.) in the Greenhouses Located
in Yeosu, Jeonnam Province, Korea
Je Yong Jeong, Chang Jeon Kim, Hye Yeon Mun, and
Hyang Burm Lee
Chang Jeon Kim1, Hye Yeon Mun1, Hyo-Suk Choi2,
Young-Hyuk Lee2, Hye-Ok Yun2, Yu-Kon Kim2, and
Hyang Burm Lee1
Division of Applied Bioscience & Biotechnology, College of
Agriculture & Life Sciences, Chonnam National University
In total, 106 isolates of endophytic fungi were isolated from
leaves and stems of red pepper (Capsicum annuum L.)
cultivated in greenhouse and open field from 2008 to 2009.
Isolation rate of the endophytes in the open fields (77%) were
higher than that in greenhouses (39%). The isolation rates in
greenhouses were 42% and 35% in environment-friendly and
conventional cultivation sites, respectively. All the isolates
were identified on the basis of their morphologies as well as
18S rDNA sequence analysis. Alternaria (62%) and Cladosporium (32%) were dorminant species in the open fields and
greenhouses, respectively. Chaetomium (15%) and Alternaria
(13%) were prevalent in the greenhouses while Cladosporium
(5%) and Chaetomium (3%) in the open fields. Thirty nine
isolates comprising 10 genera both from greenhouses and open
fields included Athrinium phaeospermum, Cercospora zebrinae.
Fusarium spp., Gibberella moniliformis, Hypoxylon howeanum,
Microascus triqonosporus, Nigrospora sp., Phaeosphaeriopsis
musae, Sordariomycetes spp., and Xylaria spp. et al.
B054
First Report of Powdery Mildew Caused by an
Erysiphe Species on Quercus phillyraeoides
Chang Jeon Kim*, Hye Yeon Mun, and Hyang Burm Lee
Division of Applied Bioscience and Biotechnology, College of
Agriculture & Life Sciences, Chonnam National University
In May 2009, a powdery mildew on Quercus phillyraeoides
A.Gray has been first observed at the campus of Chonnam
National University, Gwangju, Korea. White powdery growth
was abundant on the upper leaf surfaces. Leaf symptoms
appeared commonly white in May to October. Along with the
typical white powdery mildews, spot and/or necrotic symptoms
with irregular violet to wine red-colored surfaces were also
frequently observed on overwintered leaves. A voucher
specimen has been deposited in EML herbarium collection,
Chonnam National University (EML-QUP1). Conidia were
formed singly and the size was 36.6-42.4 (av. 36.0) µm in
length x 11.9-17.1 (av. 14.7) µm in width. Sequence analysis
by BLAST indicated that EML-QUP1 was closest to an
Erysiphe species, E. quercicola (accession no. AB292693)
with 96% sequence similarity. So far, it has been known that
Cystotheca lanestris, Erysiphe heraclei, Microsphaera alphatoides and Phyllactinia quercus cause powdery mildews on
Quercus plants. To our knowledge, this is the first report of
leaf powdery mildew caused by an Erysiphe sp. different with
E. heraclei on Q. phillyraeoides in Korea.
160
Poster Sessions
1
Division of Applied Bioscience and Biotechnology, College of
Agriculture & Life Sciences, Chonnam National University,
2
Agricultural Technology Center
Gypsophilas are grown both as garden plants and also valuable as a
cut flower in floristry to add as a filler to flower bouquets. Two
forms of gypsophilas are commonly cultivated: the annual
Gypsophila elegans, and the perennial G. paniculata. In September
of 2009, a severe wilt disease was observed on G. elegans in the
greenhouses located in Yeosu, Jeonnam province, Korea. Ten
isolates were isolated from the stems and roots of wilted gypsophila.
Three Fusarium isolates (EML-GYP1, 2 and 3) showing different
colony types were selected and studied. Sequence analyses by
BLAST indicated that two isolates of EML-GYP1 and 2 were
closest to F. proliferatum with 99% sequence similarity and another
isolate of EML-GYP3 was related to F. oxysporum with 97%
sequence similarity. Pathogenicity tests with the three isolates were
performed on G. elegans. Seven days after inoculation, similar
symptoms to those observed in the greenhouses were seen on the
inoculated plants. The causal fungus was re-isolated from the
artificial lesions fulfilling Koch's postulates. Control plants showed
no wilt symptoms. EML-GYP2 isolate was more pathogenic to the
gypsophila plants than EML-GYP1 and 3. So far, only F.
oxysporum has been known to cause wilt disease on gypsophilas in
Korea. However, this study showed that another Fusarium species
along with F. oxysporum could cause the wilt disease on gypsophila.
B056
Pantoea spp. Related to Leaf Blights on Rice in Paddy
Fields Located in Gwangyang and Naju, Jeonnam
Province, Korea
Jin Pyo Hong1, Seung Bum Kim2, and Hyang Burm Lee1
1
Division of Applied Bioscience & Biotechnology, College of
Agriculture & Life Sciences, Chonnam National University,
2
Department of Microbiology, Chungnam National University
In September 2009, leaf blights were observed on rice (Oryza
sativa L.) in paddy fields located in Gwangyang and Naju,
Jeonnam province, Korea. Early lesions began as water soaked
stripes or light brown to slightly reddish spots on upper blade
of leaves, ultimately causing leaf blights. Four strains (EMLORY1 to 4) were isolated from the leaf lesions and all the
isolates formed yellow colonies on LB medium. The strains
were identified based on the 16S rDNA sequence analyses.
Three strains of EML-ORY1, 2 and 3 belonged to genus
Pantoea and were closest to Pantoea ananatis (ATCC19321)
sharing >99% sequence similarity while EML-ORY4 strain
was mostly related to P. agglomerans (AJ233423), sharing
98.8% sequence similarity. Pathogenicity tests were performed
on 2-weeks-old rice seedlings with bacterial suspensions
containing 108 cfu/ml with 0.001% tween-20. Out of them,
EML-ORY3 showed the strongest pathogenicity to rice.
B057
Effect of Water Activity (aw) and Temperature on
Mycelial Growth of Endophytic Pestalotiopsis spp.
Hyoung Ryoul Jang, Hye Yeon Mun, Chang Jeon Kim, and
Hyang Burm Lee
Division of Applied Bioscience & Biotechnology, College of
Agriculture & Life Sciences, Chonnam National University
The effect of water activities (0.995, 0.98, 0.95 aw and control) and
temperatures (18, 23, 27 and 32°C) on mycelial growth of 6
endophytic isolates of Pestalotiopsis were investigated on V-8
juice and malt extract agar media. The six isolates (JUN-1 and 2,
QUE-1 and 2, PIN-1 and 2) were isolated from Chinese juniper
(Juniperus chinensis Linn.), oriental chestnut oak (Quercus aliena
Blume) and Japanese red pine (Pinus densiflora Sieb. et Zucc.),
respectively.The mycelial growth of different isolates of
Pestalotiopsis varied with water activity and temperature. Out of
six isolates tested, QUE-1 isolated from Q. aliena grew fastest
while PIN-2 from P. densiflora slowest. Decrease (0.95 aw) in
water activity caused a significant reduction in mycelial growth of
all the six isolates. Mycelial growths of Pestalotiopsis spp. were
highest on MEA and at 27°C. At 32°C, the growth rates of QUE-1
and 2 from Q. aliena in comparison with other isolates were higher
at 0.98 aw than other water activity levels.
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C001
Isolation and Characterization of HistamineProducing Bacteria in Fermented Fish Products in
Korea
Jin Seok Moon1*, Kyung-Ju Cho2, Seung-Joon Yang2,
Gun-Mook Yoon2, So-Young Kim3, Seung Kee Cho1,
Yu Jin Kim1, and Nam Soo Han 1
1
Department of Food Science and Technology, Research Center
for Bio Resource and Health, Chungbuk National University,
2
Chungbuk Institute of Health & Environment Research,
3
Functional Food & Nutrition Division, Department of Korean
Food Research for Globalization, National Academy of
Agricultural Science
The objective of this study was to isolate and characterize
histamine-producing microorganism in fermented fish products
in Korea, including anchovy sauce, sand lance sauce, squid
paste, clam paste, and shrimp paste. For screening of
histamine-producing bacteria, histamine detection agar (HDA)
medium was used and the multiplex PCR was done for
genomic analysis. As a result, two histamine-producing bacteria
were isolated from anchovy sauce and sand lance sauce, and
they were identified as Bacillus licheniformis and Bacillus
coagulans. The multiplex PCR analysis showed existence of
histidine decarboxylase (HDC) the chromosome of those
bacteria. At the same time, their abilities to produce biogenic
amines in histidine-containing media were confirmed by
HPLC analysis. Further application of these culture dependant/
independant methods will permit wider monitering of
histamine-producing bacteria in various fermented foods.
[Supported by the MKE and KIAT]
C002
C003
Antimicrobial Treatment of Grapes Using Sodium
Hypochlorite (NaOCl) in Winemaking and Its
Effects on Chemical and Sensory Characteristics of
Wine
Ki-Seon Yoo*, Ji Eun Kim, Hwa Young Choi,
Eun-Young Seo, Yu Jin Kim, and Nam Soo Han
Department of Food Science and Technology, Research Center
for Bioresource and Health, Chungbuk National University
The aim of this study was to evaluate the use of NaOCl as an
alternative antimicrobial compound in winemaking. Analyses
of microorganisms, chemical changes and sensory characteristics
were carried out during the fermentation periods of three
wines; non-treated, SO2-added or NaOCl-treated wines. Treatment of grapes with NaOCl reduced the initial contaminating
microbial population in red wine and this resulted in higher
growth of yeast and LAB in alcoholic and MLF periods. After
200 days incubation, the chemical analysis of NaOCl-treated
wine showed that ethanol content, redness (a*) in color,
concentrations of fruity-ester compounds were higher and the
total acidity was lower than the non-treated. In the sensory
analyses, the NaOCl-treated wine obtained significantly
(p<0.05) higher overall acceptability score (5.70) than the
SO2-added wine (4.26). These results revealed that NaOCl
acted as an alternative of SO2 in winemaking giving an
inhibitory effect against the contaminating microorganisms and
it might cause an environmental change toward a better wine
fermentation condition for yeast and LAB.
[This study was supported by grants from TDPAF and NRF]
C004
Microbial Community Associated with Internal
Organs of Snow-Crabs
Identification of Antibiotics against Pathogenic
Fungi from Bacillus subtilis
Jin-Uk Lee1*, Na-Yeon Kim2, Gyeong-Jin Jo2,
Jeoung-Hyun Hong1, Young-Eun Lee1, Nyun-Ho Park1,
Choong-Gon Kim 1, and Jong-Shik Kim1
1
Gyeongbuk Institute for Marine Bioindustry, 2Yeungnam
University
Haemin Kim*, Yong Hoon Lee, Bong-Sik Yun,
Byung-Taek Oh, Kui-Jae Lee, and Jong-Chan Chae
Division of Biotechnology, College of Environmental &
Bioresource Sciences, Chonbuk National University
The purpose of this study was to analyze microbial community
associated with the organs of Uljin snow-crab(Chionoecetes
opilio), Red tanner crab(Chionoecetes japonicus) and NeodoDaege(Chionoecetes sp.). To our knowledge, there have yet
studied what kinds of microbes are associated with their organs.
Here, we have been analyzing the microorganisms of their
organs using 6 different media for isolation. Microbes from
organs of three crabs have been isolated (369 isolates) and
identified by sequence analysis of 16S rRNA and ITS gene.
From Uljin snow-crab, Acinetobacter, Stenotrophomonas and
Pseudomonas were isolated mainly, and from a red tanner crab,
Psychrobacter and Acinetobacter and from a Neodo-Daege
snow-crab, Rhodococcus, Leifsonia and Bacillus. In addition,
antibiotic susceptibility tests showed that Lactobacillus,
Pseudomonas and Enterobacter from Uljin snow-crab and
Claribacter, Psychrobacter, Lactobacillus from red tanner crab
were resistant to tested all nine antibiotics, mainly isolates
from Uljin snow-crab were high resistant to penicillin,
erythromycin, vancomycin and ampicillin.
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Poster Sessions
A halotolerent bacterial strain was isolated from Suncheon Bay
exhibiting strong antagonistic activity against various plant
pathogenic fungi such as Alternaria alternate, Aspergillus
niger, Botrytis cinerea, Collectotrichum acutatum, Collectotrichum coccodes, Diaphorina citri, Penicillium digitatum, and
Penicillium italicum. Additionally, in vivo antagonistic assay
on postharvest peppers showed inhibition activity against C.
acutatum. The strain was identified as a Bacillus subtilis based
on 16S rDNA sequences and produced antifungal compounds
on Luria-Bertani media at 30°C. The crude compounds were
precipitated by adjusting pH to 2.0 with 6 mol of HCl in the
cell free culture broth, which were extracted with methanol.
They were classified as lipopeptides according to the
molecular masses by MALDI-TOF mass spectrometry. The
estimated molecular weight peaks 1,080, 1,058 and 1,485
(m/z) indicate that the compounds belong to iturin, surfactin
and fengycin families.
C005
C007
Screening for Cold-Active Protease-Producing
Bacteria from the Culture Collection of Polar
Microorganisms and Protease Characterization
*
Ha Ju Park , Dockyu Kim, Yung Mi Lee, Soon Gyu Hong,
Hong Kum Lee, and Joung Han Yim
Polar BioCenter, Korea Polar Research Institute
KOPRI has assembled a culture collection of cold-adapted
bacteria from the Arctic and Antarctic. To identify excellent
protease-producers among the proteolytic bacterial collection
(874 strains), 78 strains were selected according to their
relative activities and were subsequently re-examined for their
extracellular protease activity on ZoBell/skim milk plates. This
screening method permitted the selection of a small group of
15 cold-adapted bacteria, belonging to the genus Pseudoalteromonas or Flavobacterium, that showed proteolytic activities at
5-15°C. The cold-active proteases from these strains were
classified into four categories according to the extent of
enzymatic inhibition by a class-specific protease inhibitor.
Since highly active and cold-adapted proteases have the
potential for commercial enzyme development, the bacteria
selected in this work are studied as a valuable natural source of
new proteases. At present, a protease from one strain (KOPRI
21717), which is highly active at low temperatures and
resistant to protein-denaturing SDS, is being studied for future
application in detergent.
Development of Functional Probiotics for Prevention of Dental Caries
Donghwan Kim*, Yesl Sin, Kijong Yu, and Hongik Kim
R&D Division, VITABIO, Inc.
Dental caries is one of the most common oral diseases in
human and Streptococcus mutans is its major causative
microorganism. Glucosyltransferase (GTase) secreted by S.
mutans produces insoluble glucan that plays an important role
to form the biofilm. S. mutans under the biofilm demineralizes
of tooth enamel by acids, finally induces dental caries.
Therefore, inhibition of S. mutans growth and GTase activity
will be effective in prevention of dental caries at the early stage
of biofilm formation. In this study, 6 probiotic bacteria were
isolated from kimchi and human feces. These strains had more
potent antimicrobial activity against S. mutans compared to
other reference strains. They effectively inhibited the formation
of biofilm, in vitro. These probiotics were identified as
Lactobacillus plantarum, Weissella confusa and W. cibaria by
phylogenetic analysis using 16S rDNA. In addition, these
isolates were resistant to gastric acidity and bile, and possessed
antimicrobial activity against E. coli and Salmonella
typhimurium. Therefore, it is expected that these probiotics
may prevent dental caries and also show good probiotic effects
through oral administration.
[This work was supported by a grant to KOPRI (PE09050).]
C006
C008
Silver and Zinc Coated Silica as a Preservative for
Personal Care Product
Study on Regarding a Hypha Culture Characteristic of a Naematoloma sublateritium
Hyun Sang Lee1*, Yun Jeong Kim1, Ji Hyun Son1,
Neri Geum2, and Jong Woo Chun1,2
1
R&D Center, ACT Co., Ltd., 2Department of Chemistry,
Dankook University
Jongwoon Choi1*, Jihong Kim2, Dusik Jeon1,
Gilhwa Jeong1, Jiyoung Choi1, Sanghoon Chung2,
Sunga Choi3, and Sangjun Sim1
1
Forest Research Institute of Gangwon-do, 2Department of
Forest Management, Kangwon National University,
3
Department of Life Science, Hallym University
Silver and zinc ion coated silica (ACTOSIL) was synthesized
for the purpose of using a preservative for personal care
product. To evaluate the antimicrobial activity of ACTOSIL,
MIC tests were performed against various microorganisms.
The MIC values of ACTOSIL against various bacteria and
fungi were 12.5 to 100 ppm and 100 to 500 ppm respectively.
Challenge test were conducted to test the antimicrobial effect
of ACTOSIL in various personal care products. At the
concentration of 15 ppm, ACTOSIL meets the recommendation
of CTFA microbiological guideline. Furthermore, the result of
MTT assay with ACTOSIL showed low cell cytotoxicity. As
shown these results, ACTOSIL has a potential to use a safe
preservative for personal care products.
This study executed it in order to analyze a characteristic in a
hypha culture of a Naematoloma sublateritium. It was excellent,
and a GP medium, a YMA medium was investigated, but
existing other study to a PDA medium it was investigated.
Suitable temperature and provisional results regarding ph of a
Naematoloma sublateritium, proper temperature appeared with
25 degrees, and proper ph was investigated to 6.5~7.0.
This study showed to excellent hypha growing at Glucose in
circular the carbon which was suitable for the third, hypha
growing of a Naematoloma sublateritium provisional results
carbon circle regarding a nitrogen circle, and cultures of a
Naematoloma sublateritium looked at malt extract in case of
organic nitrogen circles.
This study looked in a phosphoric acid circle to affect the
fourth, a mycelium of a Naematoloma sublateritium to growth
and development. Also, If this study added it was investigated
p-aminobenzoic acid in a vitamin circle so that hypha growing
was comparatively good.
[This paper consisted by assistances of a technology development assignment scientific forest.
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C009
Co-Culturing Bacillus lichinoformis with Clostridium
tyrobutyricum for Production of Butyric Acid from
Sucrose
Mohammed Dwidar1,2*, Se-il Kim1, Jae-Yeon Park1,
Byung-Seung Jeon1, Youngsoon Um1, and Byoung-In Sang1,2
1
Korea Institute of Science and Technology, 2University of
Science and Technology
An isolated strain of Bacillus lichinoformis was used in a coculture with Clostridium tyrobutyricum ATCC 25755 in serum
bottles containing RCM supplemented with sucrose as the sole
carbon source for the production of butyric acid. Although
C.tyrobutyricum alone is unable to use sucrose as a carbon
source, this co-culture was able to use sucrose and to produce
butyric acid up to 10 g/L which is even higher than that
produced from C.tyrobutyricum alone in serum bottles when
glucose is used as a carbon source under the same conditions.
B.lichinoformis alone did not show any butyric acid production,
implying that B.lichinoformis degrades sucrose into monosaccharides by specific enzymes and then C.tyrobutyricum
uses them for butyric acid production. Butyric acid was the
main product in this co-culture. Also, the final acetic acid
concentration in the medium was significantly lower than that
produced from C.tyrobutyricum alone when glucose is used as
a carbon source. In addition, this co-culture could grow in the
RCM broth in serum bottles without initial purging with inert
gas for providing anaerobic conditions.
C011
Use of Bacteria Isolated from Dok-do Island for
Improving Compressive Strength in Cement-Sand
mortar
Sung-Jin Park1*, Sung-Tae Kim2, Wha-Jung Kim2, and
Sa-Youl Ghim1
1
School of Life Sciences, Kyungpook National University,
2
School of Architecture & Architectural Engineering,
Kyungpook National University
This study shows an application of microbiologically induced
carbonate precipitate for strength improvement of cement-sand
mortar. Nine calcium carbonate forming bacteria (CFB) were
isolated from Dok-do and partially identified by performing
sequence analysis of the 16s rRNA gene. Crystal aggregates
were apparent in the bacterial colonies grown on an agar
medium. The crystals on urea-CaCl2 agar medium were
captured by a Zentech digital camera equipped with a
stereomicroscope (Sw 804425; Samwon, Korea). These strains
showed strain specific CaCO3 precipitation on urea-CaCl2
medium. To verify an effect of improved compressive strength
in cement-sand mortar, the CFB were inoculated in cementsand mortar.
[Supported by grants from KOSEF and KRF]
[Supported by grants from SK energy]
C010
C012
Synthesis of Chalcogenide Ternary and Quaternary
Nanotubes through Directed Compositional Alterations of Bacterial As-S Nanotubes
Purification of Determinants of Ochrobactrum
lupini KUDC1013 Inducing Systemic Resistance
against Soft-Rot Pathogen in Tobacco
Shenghua Jiang1*, Fang Liu2, Min-Gyu Kim3, Jae-Hong Lim2,
Kun-Jae Lee4, Yong-Ho Choa4, Kyung Song5, Michael J. Sadowsky6,
Wilfred Chen2, Nosang V. Myung2, and Hor-Gil Hur1,7
Marilyn Sumayo1*, Mi-Seon Hahm1, Ye-ji Hwang1,
Choong-Min Ryu2, and Sa-Youl Ghim1
1
School of Life Sciences, Kyungpook National University,
2
Laboratory of Microbial Genomics, Systems Microbiology
Research Center, Korea Research Institute of Bioscience and
Biotechnology
1
Department of Environmental Science and Engineering, Gwangju
Institute of Science and Technology, 2Department of Chemical and
Environmental Engineering, University of California at Riverside,
3
Pohang Accelerator Laboratory, 4Department of Fine Chemical
Engineering, Hanyang University, 5Division of Electron Microscopic
Research, Korea Basic Science Institute, 6Department of Soil, Water, and
Climate; and BioTechnology Institute, University of Minnesota,
7
International Environmental Research Center, Gwangju Institute of
Science and Technology
Eco-friendly biological syntheses of nanomaterials have been
recently introduced as alternative protocols for the conventional
chemical routes. Previously we reported that Shewanella sp. HN41 can produce photoactive arsenic sulfide (As-S) nanotubes by
biological reduction of As(V) and thiosulfate. In this study, we
demonstrate the synthesis of chalcogenide ternary (i.e. As-S-Se
and As-Cd-S) and quaternary (i.e. As-Cd-S-Se) composited
nanotubes through bacterial Se(IV) reduction by strain HN-41
and/or abiotic ion exchange of As-S with Cd under ambient
conditions. Microscopic observations and electrical measurements
revealed that morphology, crystal structures, and electrical properties can be significantly altered by controlling the composition.
These strategies may provide environmental friendly routes to
manufacture other nanoengineered materials and devices in costeffective matter.
[This work was supported by the 21C Frontier Microbial Genomics
and Applications Center Program (M102KK010011-08K1101-01110),
Ministry of Education, Science & Technology, Republic of Korea.]
164
Poster Sessions
Ochrobactrum lupini KUDC1013 is a plant growth promoting
rhizobacterium (PGPR) isolated from the rhizosphere of
Solanum nigrum L. plants in Dokdo island and has the
capability to induce systemic resistance (ISR) of tobacco
against the leaf soft-rot pathogen Erwinia carotovora subsp.
carotovora strain SCC1. In order to distinguish the bacterial
determinants that elicit systemic resistance, extracellular
components produced by KUDC1013 were extracted with
series of solvents, fractionated, purified and were tested for
their protective effects. ISR bioassay revealed that resistance
elicitors were retained in the cell free supernatant and was
partitioned into n-butanol. Further purification and identification analyses of the ISR elicitor from KUDC1013 will
increase the range of factors from PGPR recognized to induce
plant resistance.
C013
Base Ranking Analysis on Regarding a rDNA ITS
Part of a Naematoloma sublateritium
Jongwoon Choi1*, Dusik Jeon1, Gilhwa Jeong1,
Sanghoon Chung2, Jihong Kim2, Sunga Choi3, Sangjun
Sim1, and Jiyoung Choi1
1
Forest Research Institute of Gangwon-do, 2Department of
Forest Management, Kangwon National University,
3
Department of Life Science, Hallym University
Condition of PCR cycling repeated 30cycle to extension for
annealing/72 degrees, 90 seconds for denaturation/50 degrees,
30 seconds for 95 degrees, 30 seconds, and I performed it, and
in 95 degrees, three minutes and last extension used it the first
denaturation 72 degrees, 10 minutes. Primer ITS1 and ITS4
(White et al.)I used 1990), and analysis of PCR amplication
product is Mupid 21(COSMO Bio Co.) use, 1.I executed
electrophoresis at 508b3536p-1022garose gel (1X TAE buffer)
for 100V 30 minutes. Marker 1kb DNA ladder (Promega Co.)I
used), and I confirmed amplication product to Gel Documentation System 2000 (Bio-Rad) after I dyed it to ethidium
bromide dynamic an electric area for 20 minutes, and washing
it to a distillation number for five minutes. The Naematoloma
sublateritium isolate sympathy and phylogenetic tree analysis
results are EF530927. It is Hypholoma capnoides internal
transcribed spa. one 1277,0.I appeared to 0.0.
[This paper consisted by assistances of a technology development assignment scientific forest.
C014
Chemical Structures and Apoptosis Inducing Effect
of Diketopiperazine Disulfides Produced by
Aspergillus sp. KMD 901 Isolated from Marine
Sediment on Colon Cancer Cell Lines HCT116
Eun Ju Choi*, Jin-Soo Park, Young-Joo Kim, Ji-Hee Jung,
Hak Cheol Kwon, and Hyun Ok Yang
Korea Institute of Science and Technology, Gangneung Institute
The objective of this research was to determine chemical
structures and to estimate apoptosis inducing effect of
diketopiperazine disulfides produced by Aspergillus sp. KMD
901 isolated from marine sediment, which collected from the
East Sea of Korea. The ethyl acetate extract of strain KMD 901
exhibited the strongest cytotoxic activity on cancer cell lines.
The cytotoxic compounds were purified from the ethyl acetate
extract of Aspergillus sp. KMD 901 using reverse-phase high
performance liquid chromatography. Structures of isolated
cytotoxic compounds were elucidated diketopiperazine disulfides by means of spectroscopic analyses, including extensive
2D NMR and mass spectrometry. The diketopiperazine
disulfides induced apoptosis of HCT116 cells, characterized by
cleavage of apoptosis-related proteins using Western blotting
analysis. In vivo xenograft test, acetylapoaranotin (2) showed
antitumor effect. These results suggested that these diketopiperazine disulfides from Aspergillus sp., KMD 901, could be
a candidate for the development of antitumor agents.
[Supported by Korea Institute of Science and Technology
institutional program, Grant number, 2Z03270, Republic of
Korea.]
C015
Biotransformation of Heterocyclic Aromatic Compounds by Naphthalene Dioxygenase from Pseudomonas sp. Strain NCIB 9816-4 in Escherichia coli
Cung Nawl Thawng1* and Hor-Gil Hur1,2
1
Department of Environmental Science and Engineering,
2
International Environmental Research Center, Gwangju
Institute of Science and Technology
The naphthalene dioxygenase (NDO) from Pseudomonas sp.
strain NCIB 9816-4 is a multicomponent enzyme system that
carries out the initial step in the biodegradation of naphthalene.
This enzyme has a broad substrate range and catalyzes several
types of reactions including cis-hydroxylation, monooxygenation, and desaturation. In this study, E.coli JM109 (pDTG141)
harboring NDO gene of Pseudomonas sp. strain NCIB 9816-4
was used for whole cell reaction. Totally eight chemicals
which are heterocyclic aromatic compounds were used for this
biotransformation experiments. NDO was found to be
produced metabolites from all tested chemicals. The possible
products of each metabolite were identified by HPLC and
LC/MS analysis. Future study by LC-NMR will confirm the
structures of metabolites of each compound.
[This work was supported by a grant from the MEST/NRF to
the Environment Biotechnology National Core Research
Center (grant #: 20090091491), Korea]
C016
Characterization of a High Salt Resistant pHydroxybenzoate Hydroxylase from Chromohalobacter sp. HS-2 in E. coli
Hee Gon Kim1*, Yu Ri Park1, Wonduck Kim2, and
Si Wouk Kim1
1
Department of Environmental Engineering, BK21 Team for
Biohydrogen Production, 2Pioneer Research Center for
Controlling of Harmful Algal Bloom, Chosun University
The gene encoding p-hydroxybenzoate hydroxylase (pobA)
from moderately halophilic bacteria, Chromohalobacter sp.
HS-2, was cloned into the expression vector pCRT7-CTTOPO. The pobA gene was expressed in E. coli BL21 (DE3)
by 0.75 mM IPTG. Expression in E. coli BL21 resulted mainly
in the formation of inclusion bodies. To enhance the
production of soluble PobA protein, chaperone plasmid (dnaKdnaJ-grpE) was co-expressed with E. coli harboring pobA
gene. SDS-PAGE and Western blot analysis showed an
estimated 44 kDa protein band corresponding to the
recombinant p-hydroxybenzoate hydroxylase. The HPLC and
LC/MS analysis revealed that resting cells of E. coli BL21
harboring pobA and chaperone plasmid oxidized phydroxybenzoate to protocatechuate. The expressed PobA
protein was purified by Ni-NTA column and characterized.
The optimal pH and temperature for enzyme activity was
determined to be 9.0 and 35°C, respectively. The Vmax and Km
values were 53.8 μmol/min/mg protein and 78.9 μM. The
relative activity of PobA protein was remained 50 and 20% in
the presence of 0.5 and 1M NaCl, respectively.
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C017
C019
The Isolation and Characterization of Antifungal
Compounds from Soil Bacteria
Ke Dong1*, Hyun-Ju Lee2, Sung-Kee Kim2, Jaisoo Kim1,
and Sang-Seob Lee1
1
Kyonggi University, 2GyeonggiDo Agricultural Research &
Extension Services
A plant fungal pathogen, Fusarium oxysporum (Fox) is the
causal agent of root rot or wilt diseases in several plant species,
including some cash crops. We want to extract some antifungal
compounds against Fox from soil bacteria. At the present, we
screened 242 strains from 42 species, and the strain 2252-010
showed the highest antifungal activity. The strain 2252-010
was identified as Psedomonas chlororaphis based on 16S
rDNA sequences. The strain 2252-010 showed efficient antifungal activities on all of the five different pathogenic strains
of Fox. The growth conditions of the strain 2252-010 for
antifungal compound production on potato dextrose agar
(PDA) plate are pH 7.0, 28°C and 3 days incubation. We are
going to extract, purify and characterize the antifungal
compound. If the compound has an efficient antifungal and
economical performance, we may use it as a novel, environmental-friendly antifungal agent.
Lactobacillus reuteri Probio-16 Isolated from Pig
Feces Inhibits Porcine Rotavirus and Enteric
Bacterial Pathogens
1*
1
1
Byeong Joo Seo , Mi Ran Mun , V. J Rejish Kumar ,
Chul-Joong Kim2, Insun Lee1, Young-Hyo Chang3,
Jeongheui Lim1, and Yong-Ha Park1
1
Department of Applied Microbiology and Biotechnology,
Yeungnam University, 2Collage of Veterinary Medicine,
Chungnam National University, 3Korean Research Institute of
Bioscience and Biotechnology
Bacterial strain Probio-16 was isolated from the swine
excrements and characterized by morphology and biochemical
characteristics. The strain was further identified by 16S rRNA
gene sequencing and phylogeneitc analysis. The strain was
tested for antiviral activity by porcine rotavirus inhibition in
vitro in African green monkey epithelial cell line TF-104.
Phenotypically and through 16S rRNA gene sequences,
Probio-16 was identified and named as Lactobacillus reuteri
Probio-16. This strain was resistant to pH 2.0, 5% bile acids
and exhibited antimicrobial activity against all the thirteen
enteric bacterial pathogens tested. Probio-16 supernatant
inhibited porcine rotavirus in vitro in TF-104 cell lines. Except
for erythromycin and penicillin G at a concentration of 4 µg/ml,
Probio-16 showed resistance to all other thirteen antibiotics
tested. This study indicates L. reuteri Probio-16 as a novel
strain with its tolerance to low pH and bile, antimicrobial
activity, antibiotic resistance and antiviral activity against
rotavirus, and an ideal probiotic candidate for animal and
human application after the proper in vivo experiments.
[Supported by the grants from Yeungnam University]
Poster Sessions
Eunyoung Seo* and Nam Soo Han
Chungbuk National University
Leuconostoc sp. is a dominant lactic acid bacterium in kimchi
and it catabolizes glucose, sucrose, and fructose producing
lactate, dextran, and mannitol, respectively. The aim of this
study was to investigate the protein expression patterns in L.
mesenteroides ATCC8293 in various carbon sources (glucose,
sucrose, and fructose). For this, L. mesenteroides was cultured
in MRS medium containing 2% of reach sugars and proteomic
analysis was performed using LC-MS. As results, about 600
proteins were isolated in the glucose-medium and the
distinctive proteins expressed more than two folds in the
sucrose-medium were identified as glycosidase, ABC-type
transport system (2 types), sugar kinase, ribosomal proteins
(two types), and heat shock proteins. The protein expression
pattern in the fructose-medium was generally same with that of
sucrose. The most highly expressed proteins in glucosemedium were enzymes or regulatory proteins involved in the
central metabolic pathway, DNA replication and protein
expression, and they were mainly house-keeping proteins, as
expected. This information will be used to construct the sugarinducible expression vector system or the in silico metabolic
pathway.
C020
C018
166
Proteomic Expression Analysis of Leuconostoc
mesenteroides Subsp. Mesenteroides ATCC 8293 in
Glucose and Sucrose and Fructose Medium
Isolation of Compounds Inhibiting Growth of
Pathogenic Fungi
Ah-Ra Goh*, Yi-Na Kim, Hyun-Gook Hwang, and
Sang Ki Choi
Department of Biological Sciences, Sunchon National University
Many pathogenic fungi tend to acquire resistance to the
antifungals although a number of antifungal drug has been
developed. Thus it is necessary to develop novel drug based on
new target proteins in fungi. To identify substances that inhibit
growth of pathogenic fungi. we attempted to screen appr. 6,800
core chemicals which were provided from Korea Chemical
bank. six hundreds eighty compounds inhibited by 80% at
4.5 μg/ml the growth of Candida grabrata, Cryptococcus
neoformans and Aspergillus niger. Finally we selected five
compounds which inhibited the growth of the pathogenic fungi
at about 0.45 μg/ml significantly and tested further. The
chemicals inhibited the growth of C. glabrata proportionally as
concentration increased from 2 μg/ml to 32 μglml. MIC of a
compound for C. albicans, C. glabrata, C. lusitaniae and C.
tropicalis was each 16, 0.5, 0.06, and 0.25 respectively, which
is similar with that of itracoazole and ketoconazole. We are
under investigation how these compounds influence fungal
growth.
C021
Optimization of Antibacterial Activity of Perilla
frutescens var. acuta Leaf against Pseudomonas
aeruginosa Using Evolutionary Operation-Factorial
Design Technique
Ung Kyu Choi*, HanBoreum Lee, Hee Sun Bae, and
Dae Hyun Kim
Pohang Center for Evaluation of Biomaterials
This study was under taken to optimize the extraction condition
using evolutionary operation-factorial (EVOP) design technique
to elicit the antibacterial activity of Perilla frutescens var.
acuta leaf against Pseudomonas aeruginosa. Higher antibacterial activity was achieved at higher extraction temperature
and in a longer extraction time. Antibacterial activity was not
affected by differentiation of the ethanol concentration in the
extraction solvent. The maximum antibacterial activity of
ethanolic extract of P. frutescens leaf against P. aeruginosa
determined by the EVOP factorial technique was obtained at
80°C (R = -0.8000**) extraction temperature, 26 h (R = -0.73**)
extraction time, and 50% (R= -0.075) ethanol concentration.
The population of P. aeruginosa also decreased from 6.660 log
CFU/ml in the initial set to 4.060 log CFU/ml in the third set.
Also the scanning electronic microscopic study of the ethanolic
extract of P. frutescens revealed potential detrimental effect on
the morphology of P. aeruginosa.
C022
C023
The Melanogenesis Activity on Fermented Extract
with Lactobacilli
Min Sik Kwon*, Dae-Myoung Yun, Tae-Young Yoon, and
Gyoseon Yeom
Skin Science Laboratory, R&D Center, iPEERES Cosmetics
LTD.
Previous work, we identified tyrosinase activity of Ephedra
sinica and so we should investigate melanogenesis inhibition
and cytoxicity of fermented Ephedra extract on melanoma
B16F10 cell. And we expect that fermented extract has lower
cytoxicity than non-fermented extract and it has little
whitening effect in skin. Ephedra was extracted with 70 w/v%
ethanol and extract was fermented using lactobacilli such as
Lactobacillus helveticus KCTC 3545. The cytoxicity was
measured by MTT(tetrazolium salt 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay and skin whitening was evaluated by melanogenesis assay. Cell viability of
fermented extract was higher than non-fermented extract. The
melanogenesis inhibitory activity of fermented extract was the
highest among those of various extracts. Therefore, this study
suggests that fermented extract may be useful as whitening
agent and irritation-reduction agent in the skin.
C024
Comparison of Skin Penetration on Fermentation
with Lactobacilli
Inhibitory Effects of Broccoli Extract on Quorum
Sensing of Escherichia coli O157:H7
Dae-Myoung Yun*, Tae-Young Yoon, and Gyoseon Yeom
iPEERES, Skin Science Laboratory
Kang-Mu Lee1*, Jeesun Lim2, Mi Young Yoon1,
Yongjin Park1, Wasimul B. Chowdhury1, Sungsu Park2,
and Sang Sun Yoon1
1
Department of Microbiology, College of Medicine, Yonsei
University, 2Department of Chemistry and Nano Sciences, Ewha
Womans University
Effective material is to be ferment, nutritive component
increase and particle of component become smaller. Therefore
it is evaluated penetration in skin. We should identify
penetration rate (PR) of Ephedra (Ma huang) extract by
fermentation with lactobacilli. In the work, Ephedra was
extracted with 70% w/v% ethanol (E) and extract was
fermented using lactobacilli such as Lactobacillus helveticus
KCTC 3545 (H) and Lactobacillus plantarum KCTC 3104 (P).
Lactobacilli were incubated in appropriated condition and
activities were measured at OD560 by ELISA reader.For
confirm penetration into skin, we used Franz diffusion system.
We prepared time by receptor-sample (10, 30, 60, 120, 240
min) and quantified amount by HPLC at each 10 v/v% extract
solution. As a result, PR of H and P were higher than E. Also,
we measured reactive activities of tyrosine-tyrosinase and LDOPA-tyrosinase in H and P. Although fermented extract is
not superior to ethanol extract, we verified dose dependent
inhibitory activities.We will make clear optimizing concentration of lactobacilli at once and we eagerly look forward to
tyrosinase inhibition and distinguished penetration in skin.
Quorum sensing is a key process for the production of
virulence determinants in pathogenic bacteria. In this study, we
investigated the effects of BE on quorum sensing (QS) and
expression of QS-controlled virulence genes in Escherichia
coli O157:H7. In assays using Chromobacterium violaceum
CV026 and Vibro harveyi BB170, the BE suppressed the
production of violacein and autoinducer-2, respectively in a
does-dependent manner. Quantitative real-time PCR and GFPpromoter fusion analysis indicated the transcriptional level of
many QS-regulated virulence genes in E. coli O157:H7 was
decreased in the presence of BE. Furthermore, C. elegans fed
on E. coli O157:H7 in the presence of BE survived longer than
those fed solely on the pathogenic bacteria. These data suggest
that the BE exerts inhibitory effects on QS-associated
virulence in E. coli O157:H7 and thus has potential to be
developed as an anti-infective agent.
[This study was supported by Yonsei University College of
Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology, 7-2009-0524 to SSY.]
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C025
Identification of a Gene Encoding Endoglucanase
That Belongs to Glycosyl Hydrolase Family 45 from
the Brown-Rot Basidiomycete Fomitopsis palustris
Ju-Hee Cha* and Chang-Jun Cha
Microbial Biotechnology Lab, Department of Biotechnology
(BK21-program), Chung-Ang University
The brown-rot basidiomycete Fomitopsis palustris is known to
degrade lignocellulosic materials and produce the three major
cellulases such as endoglucanases, exoglucanases and βglucosidase. In this study, we identified a gene encoding
endoglucanase (EG) that belongs to glycoside hydrolase (GH)
family 45 from the fungus. mRNA was extracted from the
fungus grown on a medium containing 2.0% Avicel and cDNA
was synthesized from mRNA by reverse transcription. About a
650 bp fragment was obtained by 3’-RACE (Rapid Amplification of cDNA Ends) and sequenced. After the 5’-RACE,
the full-length cDNA was obtained. The cDNA fragment
consisted of 621bp nucleotides, including an open reading
frame of 206 amino acid residues. Analysis of the deduced
amino acid sequence of the fragment revealed that the protein
has the highest sequence similarity to EG from Phanerochaete
chrysosporium (71%) which belongs to the GH family 45.
Functional studies are currently in progress to evaluate the
enzyme for industrial applications.
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Poster Sessions
C026
Characterization of a Novel β-Glucosidase
Transforming Ginsenoside Rb1 to rare Gypenoside
XVII and Gypenoside LXXV from Terrabacter
ginsenosidimutans sp. nov.
Song-Gun Kim* and Dong-Shan An
Korea Research Institute of Bioscience and Biotechnology
A new β-glucosidase, which catalyzes the conversion of
ginsenoside Rb1 to more pharmacologically active rare
ginsenosides gypenoside XVII, gypenoside LXXV, was
characterized and its gene bgpA (1947 bp) was cloned in
Escherichia coli from a novel Terrabacter ginsenosidimutans
Gsoil 3082T. BLAST search using the bgpA sequence revealed
significant homology to family 3 glycoside hydrolases. The βglucosidase expressed in E. coli showed the apparent Km and
Vmax values of 4.2mM and 100.6 μmol•min-1•mg protein-1
against p-nitrophenyl- β-D-glucopyranoside. Molecular weight
was estimated as 70,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The enzyme could
hydrolyze the two glucose moieties from the C-3 position and
the outer glucose from the C-20 site of ginsenoside Rb1. These
cleavages occurred in a defined order, with the outer glucose of
C-3 cleaved first, followed by the inner glucose of C-3, and
finally the outer glucose was cleaved from the C-20 site.
Therefore, BgpA converts selectively ginsenoside Rb1 to
gypenoside XVII to gypenoside LXXV to C-K.
D001
D003
The yiiR Gene in Escherichia coli O157:H7 Regulates
O-Antigen Synthesis
Role of yjhK Gene on Pathogenicity of Salmonella
typhimurium χ3339
Jeesun Lim*, Min Yoon, Yidan Cui, Kang-Mu Lee,
Seong-Won Nam, and Sungsu Park
Ewha Womans University
Junghwa Kang1*, Saehun Kim1, Jeesun Lim2,
Kangmu Lee2, and Sungsu Park2
1
Division of Food Science, Korea University, 2Department of
Chemistry and Nano Sciences (BK21), Ewha Womans University
It is known that the O-antigen, which consists of repeating
oligosaccharide subunits, plays crucial roles in the hostmicrobe interaction in Escherichia coli O157:H7. However,
little information is available with regard to the regulation of
O-antigen synthesis. In order to investigate the regulation
mechanism, we screened out genes regulating the O-antigen
synthesis using Caenorhabditis elegans, a host model for E.
coli O157:H7 infection. Among about 900 random mutants, a
yiiR mutant was shown to have reduced virulence against C.
elegans. The microarray analysis showed that the expression of
O-antigen synthesis operon in the mutant increased over twofold than the wild type. In agglutination assay, the yiiR mutant
formed 40% less agglutination with O157 antiserum than its
wild type. In addition, atomic force microscope (AFM) showed
that yiiR mutant had shorter chain than the wild type. These
results suggest that the yiiR gene play an important role in the
regulation of O-antigen synthesis.
It was shown that C. elegans was killed by Salmonella enterica
serovars through the persistent infection in the intestine. In this
study, about 1,000 S. typhimurium mutants were constructed
through random mutagenesis and fed into the nematodes in
order to screen out mutants showing reduced pathogenicity to
C. elegans. The yhjK mutant killed less nematodes than its
wild-type χ3339. It is known that the yhjK gene encodes a
phosphodiesterase, but its role is unknown. 2D proteome
analysis showed that the mutant produced 50% less thiolperoxidase (Tpx), compared to χ3339. The yhjK mutant also
survived less in the macrophage than χ3339. The mutant was
less tolerant to oxidative stress exerted by hydrogen peroxide,
compared to the wild-type strain. Conclusively, these results
suggest that the phosphodiesterase plays an important role in
protecting the pathogen from its host innate immunity.
[This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
Government (MEST) (Basic Science Research Program R012008-000-20593-0).]
D002
D004
The Nematode Caenorhabditis elegans Can be
Killed by Escherichia coli O157:H7 Through
Infection in the Intestine
Testing Synergistic Effect of Patulin with Antibiotics
Against Burkholderia cepacia Using Whole-Body
Fluorescent Imaging in C. elegans and Mouse
Min Youn1*, Jeesun Lim1, Kang-Mu Lee1, Younghoon Kim2,
Sae-Hun Kim2, Jang W. Yoon3, and Sungsu Park1
1
Department of Chemistry and Nano Sciences (BK21), Ewha
Womans University, 2Division of Food Science, Korea
University, 3Department of Microbiology and Research Institute
for Translational System Biomics, Chung-Ang University
College of Medicine
Seong-Won Nam*, Jeesun Lim, Min Yoon, and
Sungsu Park
Department of Chemistry and Nano Sciences (BK21), Ewha
Womans University
Caenorhaditis elegans is a model host for identifying novel
virulence factors in bacterial pathogens. A previous study1
showed that nematodes fed on enterohemorrhagic Escherichia
coli (EHEC) O157:H7 were killed within 3 hours. This killing
is mediated by a toxin secreted from the pathogen grown in the
medium containing tryptophan. In this study, we report that the
nematodes fed on E. coli O157:H7 were killed in the absence
of tryptophan and the killing was related to infection in their
intestine. Compared to the wild-type E. coli O157:H7, the
strains (ΔperA, Δtir, Δeae, ΔpO157) which have a defect on
the intimate adhesion to the intestinal cells were less
pathogenic to C. elegans. The microscopic observation showed
that the mutants were less persistent in the intestine than the
wild-type. C. elegans mutants (sek-1 and ced-3 and ced-4)
were hypersensitive to the pathogen, indicating that programmed cell death (PCD) and MARK pathway mediating
innate immunity are required for C. elegans to protect it from
bacterial infection. Conclusively, these results suggest that E.
coli O157:H7 also kill C. elegans through the infection.
In recent years, the mortality rate in the cystic fibrosis (CF)
patients infected with Burkholderia cepacia complex (Bcc)
species have been increasing because of their intrinsic
resistance against many antibiotics. Previously, we visualized
cyanide accumulated in the nematode C. elegans using a
chemosensor which display a green fluorescence upon its
binding to cyanide ion (CN-). In this study, combinations of
patulin, a QS inhibitor, and several antibiotics were tested for
antibiotic susceptibility. A combination of ceftazidime and
patulin inhibited biofilm formation of Bcc species, while
ceftazidime or patulin alone failed to inhibit biofilm formation.
In vivo efficacy test using a whole animal imaging system
indicated that the fluorescence in the mouse lung treated with
the combination was significantly lower than that in the lung
treated with only a single antibiotic. This synergistic effect was
supported by the ex vivo bacterial counting. These results
suggest that the whole-body fluorescent imaging of infected
small animals is highly useful for testing in vivo antibiotic
efficacy.
[Supported by NRF grant funded by MEST (Basic Science
Research Program R01-2008-000-20593-0).]
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D005
D007
Influence of Gene Expression by Escherichia coli
LuxS Mutant Using Microarray Analysis
Antibiotic Genotype and Resistance-Acquired
Mechanism of Vancomycin-Resistant Enterococcus
spp. (VRE) Isolated from Stool
Hyun-Ah Choi1*, Jaisoo Kim1, Yong-Hyun Cho2,
Ji-Youl Lee2, Kyong-Ran Peck3, and Sang-Seob Lee1
1
Department of Biological Engineering, Kyonggi University of
Korea, 2Department of Urology, School of Medicine, The
Catholic University of Korea, 3Division of Infectious Diseases,
Sungkyunkwan University School of Medicine
Hee-Jeong Kim*, Kang-Lim Kim, Hyo-Jin Lee,
Soo-Myung Hwang, and Kyung-Soo Chang
Department of Clinical Laboratory Science, College of Health
Sciences, Catholic University of Pusan
Bacteria can detect their own cell density by quorum-sensing
mechanism that coordinates the expression of genes. The QS
signal autoinducer-2 has beenknown to promote interspecies
signaling in a broad range of bacterial species at a critical
threshold concentration of cells. LuxS is responsible for
production of AI-2, which is involved in QS response of
Escherichia coli. In this study, we examined how expression of
virulence gene is affected by AI-2. E. coli were isolated from
patient’s catheters and luxS mutants were constructed. To
confirm mutations, we performed TLC using supernatants
extracted from mutant cultures and found mutants did not
produce detectable AI-2 through activity test. Also, to study
the roles of LuxS/AI-2 on whether this system is functional in
virulence, mutants were investigated using microarray. As a
result, LuxS-dependent signal seems to be a variety of roles at
transcriptomes level. We found that mutation of luxS altered
the gene expression directly involved in other genes associated
with virulence. Our results suggest AI-2 is an important signal
in E. coli infection of the catheter-associated urinary tract
infection.
Vancomycin-resistant Enterococcus spp. (VRE) isolated from
clinic stools were analyzed for antibiotic phenotype and
genotype. Interactions between insertion of IS1216V and
IS1542 genes and antibiotic susceptibility to teicoplanin were
investigated. Consistence of van gene in VRE cultured in broth
without vancomycin was determined. VRE strains isolated
from stools were E. faecium and E. gallinarum, but not E.
faecalis. There were 10 VanA and 1 VanB phenotypes among
11 VRE isolates which were classified as vanA genotype.
Isolated VRE showed multi-drug resistant to antibiotics such
as β-lactamase and imipenem, but sensitive to quinupristin/
dalfopristin. IS1216 gene in VRE showing sensitive
intermediate to teicoplanin was not detected or weakly
detected. Copy numbers of vanA were decreased in E.
gallinarum but not in E. faecium cultured in broth without
antibiotics. Copy numbers of vanA were immediately
recovered by addition of vancomycin. Growth time of
reference E. faecium is faster than that of reference E. faecalis
in broth with vancomycin. Reference strains cultured in broth
with vancomycin showed antibiotics resistant or intermediate
without acquisition of van genes.
[Supported by grants from MEST.]
D006
D008
Isolation Frequency and Antimicrobial Resistance
of Bacterial Pathogens Isolated from Physical
Therapeutic Instruments in Geriatric Care Hospitals
1*
2
1
Min-Joo Kim , Laurentius Jongsoon Kim , Hee-Jeong Kim ,
Hyun-Jung Jo1, and Kyung-Soo Chang1
1
Department of Clinical Laboratory Science, College of Health
Sciences, Catholic University of Pusan, 2Department of Physical
Therapy, College of Health Sciences, Catholic University of
Pusan
Though geriatric care hospitals continuously are extending,
studies on microorganisms on physical therapy equipment and
their effects on hospital-acquired infections are currently
lacking. The purpose of this study is to examine isolation
frequency and antimicrobial resistance of bacterial pathogens
isolated from physical therapeutic room in geriatric care
hospitals. Specimens for this study were collected from
physical therapy rooms in geriatric care hospitals and
examined bacteria isolation and their antimicrobial resistance
patterns. Most of the bacteria isolated from these specimens
were Acinetobacter baumannii. which showed resistance to
ceftazidime, imipenem, amikacin, ciprofloxacin, and tobramycin. They showed highest antibiotics resistance to cephalothin
and ampicillin. The isolated Enterobacteriaceae showed high
resistance to ampicillin, ampicillin/sulbactam, cefazolin, and
cefoxitin. MR-CNS was frequently isolated than MRSA. By
the results, it is possible to appearance of multi-resistance
bacteria. To control nosocomial infections in physical therapy
room in geriatric care hospitals, a continuous investigation and
hygiene control have become more important.
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Poster Sessions
Immunomodulatory Activity from Pinus palustris
Part I: Isolation and Biological Assessment In Vitro
Gyeong-Im Choe*, Byung-Jae So, Jooyea Hwang,
Hee Jung Hwang, Cheong-Up Choi, and Jae-Myung Kim
National Veterinary Research & Quarantine Service MIFAFF
Pinus palustris has been widely used as a drug and food, has
been examined bioactivities, such as antioxidant, antimicrobial
and anticancer. In the present study, the immunomodulatory
activities as cell migration, cytotoxicity and IL-6 of the various
fraction from P. palustris were investigated in vitro. The dired
plant was extracted with 80% methanol to generate its
methanol extract (PME). For some experiments, ethyl acetate
(PEA), n-butanol (PB), and aqueous (PA) were prepared in
succession from PME. Among the fractions, PEA fraction was
further separated to 8 groups by silica gel 60 G (PEA-1 ~ PEA8), and PEA-7 was identified to immunomodulatory activities.
Neutrophil migration activity of PEA-1(5 ug), PEA-2(5 ug)
and PEA-7(5 ug) fractions compared with control was 149.2%,
142.2% and 135.2%, respectively. Furthermore, PEA-7
fraction was inhibited growth of tumor cell(B16F10) and
stimulated production of IL-6 in RAW 264.7 cells. These
results indicate that the PEA-7 fraction contains potential
immunomodulatory substance and it need to research more
various study.
[This work was supported by the grants from NVRQS of
MIFAFF.]
D009
D011
Polymorphisms in VacA and CagA Affect the
Severity of Gastric Diseases
An Association Between Polymorphism in the CagA
EPIYA Motif and Development of Gastric Cancer
Sungil Jang1,2*, Kathleen R. Jones3, Cara H. Olsen4,
Young Min Joo1, Yun-Jung Yoo1,2, In-Sik Chung5,
Jeong-Heon Cha1,2, and D. Scott Merrell3
Sungil Jang , Kathleen R. Jones , Young Min Joo ,
1
3
3
4
Yun-Jung Yoo , Hak-Sung Lee , In-Sik Chung , Cara H. Olsen ,
2
2
1
Jeannette M. Whitmire , D. Scott Merrell , and Jeong-Heon Cha
1
1
Department of Oral Biology, Oral Science Research Center, BK21
project, Yonsei University College of Dentistry, 2Department of
Microbiology and Immunology, Uniformed Services University of the
Health Sciences, USA, 3Division of Gastroenterology, Department of
Internal Medicine, College of Medicine, the Catholic University of
Korea, 4Department of Preventive Medicine and Biometrics,
Uniformed Services University of the Health Sciences, USA
Department of Oral Biology, Oral Science Research Center, BK21
Project, Yonsei University College of Dentistry, 2Research Center for
Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei
University, 3Department of Microbiology and Immunology, Uniformed
Services University of the Health Sciences, USA, 4Department of
Preventive Medicine and Biometrics, Uniformed Services University of
the Health Sciences, USA, 5Division of Gastroenterology, Department of
Internal Medicine, College of Medicine, the Catholic University of Korea
Helicobacter pylori infection is regarded as a cause of diverse
gastric diseases. But why some individuals develop more severe
forms of disease remains unclear. Herein, we genotyped and
analyzed 225 Korean strains for vacA to determine if particular
genotypes varied across disease state, sex, or cagA allele. Of these
strains, 206 strains carried an s1/i1/m1 allele, 11 strains carried an
s1/i1/m2 allele, and 8 strains carried an s1/i2/m2 allele. By using
Fisher’s exact test, a statistical association between variations in the
cagA and vacA alleles was identified (P=0.0007), and by using log
linear modeling, this variation was shown to affect the severity of
disease outcome (P=0.027). Additionally, we present evidence that
variation within the m region of VacA is associated with disease
outcome (P=0.011) and the distribution of vacA alleles across sex
(P=0.008). In this population, the majority of H. pylori strains carry
the vacA s1/i1/m1 allele and the CagA EPIYA-ABD allele. These
facts may contribute to the high incidence of gastric maladies.
[This work was supported by a National Research Foundation of Korea
grant funded by the South Korean Government (grant 2009-0073957).]
1*
2
1
Helicobacter pylori causes diseases ranging from gastritis to
gastric cancer. Geographically, areas with high incidences of H.
pylori infection often overlap with areas with high incidences of
gastric cancer, which remains one of the leading causes of cancerrelated deaths worldwide. Strains of H. pylori that carry the
cytotoxin-associated gene A (cagA) are much more likely to be
associated with the development of gastric cancer. Moreover,
particular C-terminal polymorphisms in CagA vary by geography
and have been suggested to influence disease development. We
conducted a large-scale molecular epidemiologic analysis of
Korean strains and herein report a statistical link between the East
Asian CagA EPIYA-ABD genotype and the development of
gastric cancer. Characterization of a subset of the Korean isolates
showed that all strains from cancer patients expressed and
delivered phosphorylatable CagA to host cells, whereas the
presence of the cagA gene did not strictly correlate to expression
and delivery of CagA in all noncancer strains.
[This work was supported by a Korea Research Foundation grant,
which was funded by the Korean government (MOEHRD) (grant
KRF-2006-311-E00083).]
D012
D010
After Contamination of Bacteria for Soil's Quality
Improvement, DGGE and Pyrosequencing Are
Used to Check Difference of Soil
1*
1
2
Hyun Jung Kim , Jaisoo Kim , Sung-Kee Kim , and
Sang-Seob Lee1
1
Department of Biological Engineering, Kyonggi University of
Korea, 2GyeonggiDo Agricultural Research & Extension
Services
Denaturing gradient gel electrophoresis (DGGE) is to study the
diversity of soil bacteria in natural and contaminated soils.
DGGE has been widely used to investigate several patterns of
distribution of soil bacteria. Two primer sets gave a single
band when used with soil bacteria and complex fingerprints.
DGGE fingerprints were then used to compare the soil bacteria
diversity in samples obtained at different soil bacteria with
bacteria contamination soil samples. DGGE bands were each
other species so the sequence analysis compare with other
molecular technique such as phyrosequencing. We planted
cabbages in control soil and contaminated soil, and we
obsevered. as a result, growth of cabbage in contaminated soil
by bacteria is the best. The DGGE results were compared with
non-contaminated soil sample and contaminated by some
bacteria such as photosynthesis bacteria, isolated from fecal,
food and there mixture sample. In conclusion the DGGE
profiles analysis was some different pattern each samples.
Contaminated by photosynthesis bacteria sample’s had a few
band compared with other samples. And soil sample of
mixtured bacteria had a many band more than other samples.
In Vitro Screening of Antiviral Activity of Natural
Plants Against Pseudorabies Virus
Jooyea Hwang*, Byung-Jae So, Hee Jung Hwang,
Gyeong-Im Choe, Cheong-Up Choi, and Jae-Myung Kim
National Veterinary Research & Quarantine Service MIFAFF
Medicinal plants are traditionally used for the treatment of
various illness including cancers, septicemia, and infectious
diseases. Eight medicinal plants used in traditional oriental
medicine were screened for potential antiviral activity against
pseudorabies virus (PRV). The natural plants were extracted
with polarities of solvents using 80% methanol, ethyl acetate,
n-butanol, and water. The antiviral activity was determined by
means of the activity of serial dilution extracts to inhibit the
produced cytopathogenic effect (CPE). These results demonstrated that the extracts of Taraxacum mongolicum, Canavalia
gladiata, Lonicerae flos, Hovenia dulcis, and Zanthoxylum
piperitum have antiviral effects against PRV at completely
non-toxic concentration ranges from 12.5 to 50μg/ml. Furthermore, the extracts of methanol and aqueous were more
effective on the suppression of virus infection than ethyl
acetate and n-butanol extracts. There preliminary results
suggest that the examined natural plant extracts might be good
candidates in control of PRV infections.
[This work was supported by the grants from NVRQS of
MIFAFF.]
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D013
D015
Activation of Lymphocytes and Macrophage Functions by Ethyl Acetate Fraction of Rhus verniciflua
Stokes Extracts
Analysis of Quorum Sensing-Dependent Extracellular Proteases of Pseudomonas aeruginosa for
Their Involvement in Infectivity and Virulence
Jae-Myung Kim*, Gyeong-Im Choe, Jooyea Hwang,
Hee Jung Hwang, Hyun-Ok Ku, Cheong-Up Choi, and
Byung-Jae So
National Veterinary Research & Quarantine Service
Ha-Young Park*, Soo-Kyoung Kim, Yusang Choi,
Changwan Ha, Su-Jin Park, Su-jin Im, and Joon-Hee Lee
Department of Pharmacy, College of Pharmacy, Pusan National
University
Rhus verniciflua stokes (RVS) have been used as a traditional
food and medicine to enhance immune response against
infectious agents and to treat cancers. In this study, the
methanol extract of RVS and its successive n-butanol, ethyl
acetate and aqueous extracts were assessed by in vitro
neutrophil migration, spleen lymphocyte proliferation and
nitric oxide (NO) production. Thus we investigated the effects
of fractions of RVS on macrophage function including
phagocytosis and cytokine production. The ethyl acetate
fraction of RVS stimulated spleen lymphocyte proliferation,
NO production, neutrophil migration, macrophage phagocytic
activity and inhibited the viability of B16F10 mouse melanoma
cells. In addition, the ethyl acetate fraction induced production
of macrophage related cytokines such as TNF-α, IL-6 and IL1α. These results suggest that RVS ethyl acetate fraction
examined here, exhibit immunostimulatory activity, which
implicates that RVS ethyl acetate fraction may serve as a
potential source of natural immunostimulants for treatment of
some animal diseases.
Pseudomonas aeruginosa is gram-negative bacteria and a
typical opportunistic pathogen. It provokes cystic fibrosis,
microbial keratitis, and burn wound infections. The virulence
of P. aeruginosa is mainly mediated by quorum sensing (QS),
a bacterial cell-to-cell communication in bacteria. Especially,
QS of P. aeruginosa regulates several extracellular proteases
that are important in its pathogenicity. To investigate whether
the QS-dependent virulence could be mediated by these
extracellular proteases, we selected 8 candidate extracellular
proteases, protease IV, LasA, LasB, PA2939, PA1249,
PA0355, PA4171, PA3535 that are expressed in QS-dependent
manner, based on the microarray database. We cloned and
overexpressed them in P. aeruginosa and examined any
change of virulence by using insect and nematode infection
model systems, where cell free culture supernatants from the
protease-overexpressing P. aeruginosa strains were collected
and injected into Tenebrio moliter, an insect to observe their
immune response and the protease-overexpressing P. aeruginosa
strains were fed to Caenorhabditis elegans, a nematode to see
the killing or decrease of multiplication of C. elegans.
[This work was supported by the grants from NVRQS of
MIFAFF.]
D014
D016
A Diagnostic Method Based on TaqMan-PCR for
the Detection and Differentiation of Recombinant
Vaccine and Wild-Type Pseudorabies Viruses
Quorum Sensing Modulation in Pseudomonas
aeruginosa by Signals Produced by Burkholderia
vietnamiensis
Jae-Myung Kim*, Hee Jung Hwang, Jooyea Hwang,
Gyeong-Im Choe, Sang-Ho Cha, Cheong-Up Choi, and
Byung-Jae So
National Veterinary Research & Quarantine Service
Changwan Ha*, Yusang Choi, Soo-Kyoung Kim,
Ha-Young Park, Su-Jin Im, and Joon-Hee Lee
Department of Pharmacy, College of Pharmacy, Pusan National
University
Psuedorabies, also known as Aujesky’s disease, is a disease
caused by the Pseudorabies virus (PRV). Thus, recombinant
Pseudorabies vaccine containing porcine interleukin-2 (IL-2)
was developed and it is necessary to establish a detection and
differentiation of vaccine and field strains of PRV. In this
study, TaqMan real-time PCR assay was designed for
detection and differentiation of Pseudorabies recombinant
vaccine virus, from some field viruses, by comparing the
amplification of two pairs of primers corresponding to
glycoprotein B genes and porcine interteukin-2, respectively.
The assay demonstrated good analytical specificity, sensitivity
and reproducibility with coefficient of variation (CV%)
ranging from 0.15% to 1.29% and from 0.046% to 0.466% for
intra- and inter-assay variability respectively. The newly
developed real-time PCR assays could be detect vaccine
viruses from livers, brains and spleens of mouse inoculated
PRV vaccine viruses.
Pseudomonas aeruginosa is an opportunistic pathogen causing
various infections in insects, plants, and human. Quorum
sensing (QS) is a bacterial cell-to-cell communication
mechanism using small diffusible molecules. P. aeruginosa
uses acyl-homoserine lactones for QS that are common QS
signals used by most gram (-) bacteria. The genome of P.
aeruginosa codes for signal synthases and receptors, so called
LuxI-R homologues, LasI-R, RhlI-R, and QscR. QscR was
originally characterized as an orphan QS receptor that has no
cognate signal synthase gene, but recently found to share the
same signal molecule, N-3-oxododecanoyl-HSL with LasR.
Burkholderia is another important pathogenic group including
complex species, so called Burkholderia cepacia complex
(BCC). In general, BCC strains have CepR-I system for QS,
but B. vietnamiensis, a BCC strain has an additional QS system,
BviI-R, producing N-octanoyl-HSL, N-hexanoyl-HSL, Ndecanoyl-HSL, N-dodecanoyl-HSL, and N-3-oxodecanoylHSL. In this study, we show that signals produced by B.
vietnamiensis can modulate QS regulation of P. aeruginosa
through the selective activation of QscR, and as consequence,
influence on virulence of P. aeruginosa.
[This work was supported by the grants from NVRQS of
MIFAFF.]
172
Poster Sessions
D017
D019
Modulation of Quorum Sensing and Virulence by
an Unknown Function Operon, PA4350~PA4351 in
Pseudomonas aeruginosa
Analysis of Quorum Sensing Signal Production of
Pseudomonas aeruginosa Clinical Isolates from
Korean Patients
Yusang Choi*, Soo-Kyoung Kim, Changwan Ha,
Ha-Young Park, Su-Jin Im, and Joon-Hee Lee
Department of Pharmacy, College of Pharmacy, Pusan National
University
Kyung-Ju Jung*, Yusang Choi, Changwan Ha,
Ha-Young Park, Su-Jin Im, and Joon-Hee Lee
Department of Pharmacy, College of Pharmacy, Pusan National
University
Quorum sensing (QS) is a bacterial communication strategy
that enables bacteria to bear group behaviors. QS in P.
aeruginosa influences the properties related to its infectivity,
such as virulence, motility and biofilm formation. P. aeruginosa has three well-defined QS systems, LasR-I, RhlR-I and
QscR. LasI and RhlI synthesize N-3-oxododecanoyl-HSL and
N-butanoyl-HSL that bind to their cognate receptors, LasR,
QscR, and RhlR. The signal-receptor complexes activate the
transcription of their target genes. An unknown function
operon, PA4350~1 was found to influence the activities of
LasR and QscR differentially. When PA4350~1 was overexpressed, both LasR and QscR were significantly inhibited, but
to a different extent, where QscR was more strongly inhibited
than LasR. The PA4350~1 overexpression significantly
alleviated the P. aeruginosa virulence in insect and nematode
infection models, and influenced on the motility and biofilm
formation, which are crucial properties for the P. aeruginosa
infectivity. We suggest that the function of PA4350~1 operon
can reduce the virulence of P. aeruginosa through the
modulation of QS regulation.
Pseudomonas aeruginosa is an opportunistic pathogen causing
various infections on lung, urinary tract, eyes, and burn wound
sites. P. aeruginosa possesses three well-defined quorumsensing (QS) systems, LasR-I and RhlR-I, and QscR. LasR-I
consists of a signal receptor, LasR and a signal synthase, LasI
that synthesizes 3-oxo-C12-HSL. RhlI-R comprises a signal
receptor, RhlR and a signal synthase, RhlI that produces C4HSL. The third LasR-RhlR homologue, QscR has no cognate
signal synthase gene, but shares 3-oxo-C12-HSL for its signal
with LasR. To investigate the importance of QS in Pseudomonas infection in Korea, we isolated clinical strains from
Korean patients and QS signal was detected by signal diffusion
assay on solid plates containing X-gal for colorimetric
detection. Our result showed that most clinical isolates
produced QS signals at the similar level to the wild type PAO1,
but significantly lower level production was detected in about
5% of them. Here we also present some additional tests for
virulence factor production, biofilm formation, and motility in
the clinical isolates.
D018
Development of Inhibitors Against Virulence of
Pseudomonas aeruginosa
Soo-Kyoung Kim*, Yusang Choi, Ha-Young Park,
Changwan Ha, Su-Jin Im, and Joon-Hee Lee
Department of Pharmacy, College of Pharmacy, Pusan National
University
Pseudomonas aeruginosa is an opportunistic pathogen that
mainly relies on quorum sensing (QS) and biofilm formation
for its virulence. As central control of virulence, QS regulates
the expression of many genes related to virulence factor
production and biofilm enhances the persistence against
challenge by host immunity and antibiotic medication. In P.
aeruginosa, QS is mediated by two small diffusible aclyhomoserine lactones (acyl-HSLs), N-3-oxododecanoyl-HSL
(3OC12-HSL) and N-butyl-HSL (C4-HSL) which are synthesized by two signal synthases, LasI and RhlI, respectively.
These signals are recognized by three QS signal receptors,
LasR, QscR, and RhlR. To control the virulence of P.
aeruginosa, we tried to develop inhibitors against QS and
biofilm formation. A series of QS signal analogues were
synthesized based on in silico modeling analysis of QS
receptor-ligand bindings and screened for anti-QS and antibiofilm activities. Some had a significant inhibition on either
QS or biofilm formation, or both. To test the potential for an
anti-Pseudomonas agent we investigated whether these
compounds could alleviate the virulence of P. aeruginosa.
D020
Temperature Dependent Secretome Analysis of
Bacillus anthracis Sterns With CO2 and Plasmid
Cues
Sudipto Shahid*, Ji Hyun Park, Sang Hoon Kim,
Kyoung Hwa Jung, and Young Gyu Chai
Division of Molecular and Life Sciences, Hanyang University
Analysis of secreted proteins in extracellular environment by
the anthrax causative bug, Bacillus anthracis drew the key
interest for the identification of potential novel virulence
factors. Differential culture condition, presence or absence of
CO2, chromosomal and plasmid regulators modulates the
secreted proteins and were identified. Temperature is one of
the host-related signals affecting transcription of the toxin and
capsule genes. In our study a comparative proteomic approach
was employed to analyze the secreted proteins (secretomes) of
two strains of Bacillus anthracis in response to different
temperature. In minimal medium under high CO2 tension,
different temperature was introduced. A unique pattern of
protein abundance was revealed when extracellular proteins
were subjected for 2-DE. Computer assisted image analysis
followed by 2-DE facilitated the identification of unique, upor down- regulated proteins. Total 41 proteins encoded on
chromosome or pXO1 were identified by peptide mass fingerprinting. Several proteins considered as putative virulence
factors were observed represents the insights of temperature
and plasmid cues in Bacillus anthracis secretome.
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D021
D023
Lactobacillus sakei Probio 65: A Functional Probiotic for Atopic Eczema-Dermatits Syndrome
1*
1
1
Yong-Ha Park , Jeongheui Lim , Byeong Joo Seo ,
1
2
1
V. J Rejish Kumar , Young-Mi Jung , Hongik Kim ,
3
2
2
4
Yoonhwa Jeong , Han-Ki Lee , Insun Lee , Kook Hee Kang ,
5
6
5
Sung-Il Woo , Nam-Shik Kim , and Youn-Soo Hahn
1
Department of Applied Microbiology and Biotechnology, Yeungnam
University, 2Institute of Probionic, Korea Research Institute of Bioscience
and Biotechnology, 3Department of Food Science and Nutrition,
Dankook University, 4Department of Food Science and Biotechnology,
Sungkyunkwan University, 5Department of Pediatrics, College of
Medicine and Medical Research Institute, Chungbuk National University,
6
Department of Preventive Medicine, College of Medicine and Medical
Research Institute, Chungbuk National University
Lactobacillus sakei Probio 65 was isolated from kimchi, a traditional
Korean fermented food. This strain was resistant to gastric acidity, bile,
and several antibiotics and possessed antimicrobial activity against a
range of pathogenic microorganisms. To investigate whether the
probiotic activity of L. sakei probio 65 was effective for treating allergic
dermatitis, the organism was supplied to mice triggered by allergen (1chloro-2,4-dinitrobenzene). Mice that received L. sakei Probio 65
showed a more rapid recovery compared to control mice, as assessed by
visual evaluation of the severity of allergic dermatitis and levels of
immunoglobulin (Ig) E and interleukin (IL)-4. Probio 65 exhibited good
probiotic properties in vitro and in mice and was effective in reducing
allergen-induced skin inflammation through the regulation of both
elevated IgE and IL-4 in sensitized mice. Supplementation of L. sakei
Probio 65 in children with atopic eczema-dermatitis syndrome (AEDS)
resulted in substantial clinical improvement and a reduction in severity
of AEDS evidenced by a significant decrease in chemokine levels.
Evaluation of Antimicrobial Effect of Ursolic Acid
Using Antimicrobial Model System for Screening of
Anti-Caries Agent
Chun Sung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1
Department of Oral Biochemistry, School of Dentistry, Chosun
University, 2Department of Biochemistry and Molecular Biology,
Medical School, Chosun University
1
The purpose of this study was to determine the antimicrobial
activity of ursolic acid against mutans streptococci isolated
from oral cavity of Koreans. The antimicrobial activity was
evaluated by minimal inhibitory concentration (MIC),
minimum bactericidal concentration (MBC) and time-kill
curves on mutans streptococci (MS). We were tested by MTT
assay in human gingival fibroblasts (HGF) and periodontal
ligament fibroblasts (PLG). MIC90 values of ursolic acid for
Streptococcus mutans, Streptococcus sobrinus and Streptococcus downei isolated from Korean were 2 ug/ml, 4 ug/ml
and 8 ug/ml, respectively. Ursolic acid had bactericidal effect
on MS. Cytotoxicity values were 16 ug/ml on HGF and PGL
for ursolic acid. The results indicate that ursolic acid have
antimicrobial activity at mutans streptococci and can be useful
in the development of oral hygiene products for the prevention
of dental caries.
[This study was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare &
Family Affairs, Republic of Korea. (grant number: A091074)]
[Supported by grants from Ministry of Health & Welfare and
Yeungnam University]
D022
D024
Antimicrobial Effects of Methanol Extracts of
Phellodendric cortex, Coptidis rhizoma and Galla
rhois on Mutans Streptococci Isolated from Koreans
Development of Prevotella intermedia-Specific PCR
Primers Based on the P. intermedia-Specific DNA
Probe
Chun Sung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1
1
Department of Oral Biochemistry, School of Dentistry, Chosun
University, 2Department of Biochemistry and Molecular Biology,
Medical School, Chosun University
Min Jung Kim1*, Jae-Yoon Park2, and Joong-Ki Kook1
1
Department of Oral Biochemistry, School of Dentistry, Chosun
University, 2Department of Biochemistry and Molecular Biology,
Medical School, Chosun University
The purpose of this study was to determine the antimicrobial
activity of methanol-extracted Phellodendirc cortex (P. cortex),
Copitidis Rhizoma (C. Rhizoma) and Galla rhois (G. rhois)
against mutans streptococci (MS) isolated from Koreans. The
antimicrobial activity was evaluated by minimal bactericidal
concentration (MBC) against clinical strains of MS as well as
type strains of Streptococcus mutans and Streptococcus
sobrinus and time kill-curve assay on type strains of S. mutans
and S. sobrinus. MBC90 values of P. cortex and C. Rhizoma
were 1 mg/ml against clinical strains of MS isolated from
Korean. MBC90 values of G. rhois for S. mutans and S.
sobrinus isolated from Korean were 2 mg/ml and 1 mg/ml,
respectively. Methanol-extracted P. cortex, C. Rhizoma, and G.
rhois had bactericidal effect on MST. The results suggest that
methanol-extracted P. cortex, C. Rhizoma, and G. rhois could
be useful in developing of oral hygiene products.
The purpose of this study is to develop the Prevotella
intermedia-specific PCR primers based on the P. intermediaspecific DNA probe. The P. intermedia-specific DNA probe
was screened by inverted dot blot hybridization and confirmed
by Southern blot hybridization. The nucleotide sequences of
species-specific DNA probes were determined by chain
termination method. The specificity of the PCR primers was
tested against 6 clinical isolates of P. intermedia, 10 clinical
isolates of Prevotella nigrescens, and 20 type strains of oral
bacteria. The sensitivity of PCR primers was determined by
testing serial dilutions of the purified genomic DNA of P.
intermedia ATCC 49046. The data showed that the two sets of
PCR primers, Pig27-F2/Pig27-R2 and Pig27-F5/Pig27-R5 had
the species-specificity for the P. intermedia ATCC 49046. The
detection limits of four primer sets were 0.4 pg-4 pg of the
purified genomic DNA of P. intermedia ATCC 49046. These
results suggested that the two sets of PCR primer, Pig27F2/Pig27-R2 and Pig27-F5/Pig27-R5, could be useful in the
development of PCR kit for the epidemiological studies and
bacteriological diagnosis or prognosis for the endontitis and
periodontitis.
[This study was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare &
Family Affairs, Republic of Korea. (grant number: A091074)]
174
Poster Sessions
D025
Differential Gene Expression in Two Phase Variants
of Streptococcus pneumoniae
Sungkyoung Lee*, Jaehoon Lee, Yeonho Kang, Jaeyon Yu,
and Songmee Bae
Division of Bacterial Respiratory Infections, Center for
Infectious Diseases, National Institute of Health, Korea Centers
for Disease Control and Prevention
Pneumococcal invasive diseases such as pneumonia,
bacteremia, and meningitis can be the result of infection of the
bacteria from upper respiratory tract or lung to blood or
cerebrospinal fluid. Pneumococcal phase variation related with
invasion to host has been described, from transparent phase for
colonization to opaque phase for invasion. To gain more
understanding on invasive pneumococcal pathogenesis, we
analyzed the transcriptome profiles of S. pneumoniae in
opaque phase to transparent phase by microarray analysis.
Each opaque and transparent colony of S. pneumoniae D39
was collected as uniform, respectively and used for microarray
analysis. The 2,240 oligo probes were designed on the basis of
full genome data of S. pneumonaie D39. Total 148 genes with
altered transcription of opaque to transparent variants were
sorted by criteria of a >2-fold change in levels of expression
and a P value of <0.05. At least 20 genes including lacA, lacB,
lacC, aga, bgaA, and bgaC, related with carbohydrate metabolism, were highly upregulated in opaque phase. Results
suggest that some of these genes are involved in pneumococcal
pathogenesis.
D027
Antiviral Activity Against Human Influenza Virus
H1N1 (PR8) From Aqueous Extract of Fritillaria
thunbergii
Nguyen Dinh Van*, Jae-Yu Choi, Youn-Dong Cho,
Jang-Hyun Lee, Hyun-Dong Paik, and Young Bong Kim
College of Animal Bioscience and Technology, Konkuk University
More than fifty extract samples from different Korean
traditional herbal remedies were screened for the antiviral
activity against the human influenza virus A/Puerto Rico/8/34
H1N1 (PR8) and toxicity by the in vitro system on MDCK
cells. The medical dried plants were extracted by two methods,
using methanol solvent at room temperature or boiling in water.
The extracted solutions were filtered, evaporated and then
evaluated the antiviral ability against H1N1 (PR8). Among the
different potential herbal remedies, Fritillaria thunbergii (F.
thunbergii) extract from the boiling water method showed
meaningful antiviral ability with high selectivity index (SI)
value, nearly 64, compared with Tamiflu? (Oseltamivir,
Roche) with 96 SI. To confirm the antiviral activity of F.
thunbergii extract, we tested it in ovo system. From this
research; our findings indicated that F. thunbergii has a good
inhibition effect on replication of the human influenza virus
H1N1 with low toxicity. This new compound is expected to be
a new drug candidate for anti-influenza therapeutics medicines
in the future.
[Supported by a grant from Korea CDC.]
D026
D028
Global Transcriptome Analysis of Streptococcus
pneumoniae Response to Hydrogen Peroxide
The Impact of Storage Duration on Bacillus
anthracis H9401 Spores
Songmee Bae*, Sungkyoung Lee, Jaehoon Lee,
Jaeyon Yu, and Yeonho Kang
Division of Bacterial Respiratory Infections, Center for
Infectious Diseases, National Institute of Health, Korea Centers
for Disease Control and Prevention
Hyun-Jung Kim*, Jeong-Hoon Chun, Hong-Mi Kim,
Gi-Eun Rhie, Cheon-Kwon Yoo, and Hee-Bok Oh
Division of High-Risk Pathogen Research, Center for Infectious
Disease, National Institute of Health, KCDC
Oxidative stress is a crucial stimulus for pathogenic bacteria
because pathogens are exposed to reactive oxygen species
produced by the immune response of the host during infection.
We designed S. pneumoniae D39 microarray chips and
investigated the dynamics of gene expression profiles during
the response of S. pneumonia to oxidative stress by 2mM
hydrogen peroxide.
As a result, 128 and 186 genes showed significant increases
and decreases, respectively, based on criteria of a greater than
2-fold change in mRNA levels and a P value of less than 0.05.
From the analysis of their cellular role of total 314 genes by
EMPAS program, the data indicate that the oxidative response
includes the induction of genes involved in virulence, repair
systems, and carbohydrate metabolisms, etc. Particularly,
expression of genes encoding chaperone such as dnaK, dnaJ,
and grpE and a gene encoding an alcohol dehydrogenase were
upregulated upon early 5 min exposure of hydrogen peroxide.
Herein, we showed the transcriptome profiles of the cellular
response of S. pneumoniae to hydrogen-peroxide induced
oxidative stress.
[Supported by a grant from Korea CDC.]
We has been producing and storing Bacillus anthracis H9401
strain spores in support of vaccine and therapeutic testing
programs against anthrax for over ten years. Spore suspensions
are either stored at 2-8°C and ≤-70°C in sterile water with 1%
phenol, 1% pheonol-10% glycerol, 10% glycerol, 70% ethanol
or 0.1% gelatin-PBS buffer. The goal of this research is to
determine the most appropriate long-term storage conditions of
Bacillus anthracis spore suspensions by examining the impact
of storage duration, media, and temperature on spore
characteristics. To date, we have studied spores placed into
storage at regular intervals beginning August 2007 through
July 2009. Enumeration of spore stored at 2-8°C in all storage
medias were consistently within ± 0.09 log of initial
concentrations(CFU/ml). Five spore lots stored for 24 months
have been tested via guinea pig lethal dose (LD50) with i.m.
injection and results indicated no change in the LD50 value at
2-8°C. These initial results led to design and implementation of
a two year controlled study to examine the impact of storage
media, temperature, and duration on viable spore counts and
virulence.
[Supported by grants from KCDC]
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D029
D031
Immune Responses of Human Papillomavirus 16/18
Bivalent DNA Vaccine; AcHERV-HP16L1 and
AcHERV-HP18L1
Quorum Sensing of Pseudomonas aeruginosa Is
Abnormally Controlled Under Anaerobic Growth
Condition
Hee-Jung Lee1*, Yoon-Ki Hur1, Jae-Yu Choi1,
Hee-Jung Cho2, Yu-Kyung Oh2, and Young Bong Kim1
1
Department of Ainmal Biotechnology, Konkuk Univeristy,
2
School of Pharmacy, Seoul National University
YongJin Park*, Mi Young Yoon, Kang-Mu Lee, and
Sang Sun Yoon
Department of Microbiology, College of Medicine, Yonsei
University
Vaccination against the most common oncogenic human
papillomavirus (HPV) types, HPV 16 and HPV 18, could
prevent development of up to 70% of cervical cancers
worldwide. We developed a novel DNA vaccine for HPV; a
recombinant baculovirus bearing human endogenous retrovirus
(HERV) envelope protein, which cannot replicate in mammals,
was used as a nano carrier for HPV-L1 DNA vaccines
(AcHERV-HP16L1, AcHERV-HP18L1). For in vivo test, mice
were injected intramuscularly with 107 particles of the
constructs, with two boosts at 2-week intervals. Compared
with Cervarix?, or Gardasil? (1/20 of human dose), the
AcHERV-HP16/18L1 immunized mice showed similar high
levels of humoral immunity in IgG/IgA and elicited strong
HPV16/18L1-specific CD8+ T cell immunity. Therefore, we
suggest that AcHERV-HPV16L1 and AcHERV-HP18L1
vaccine can be further developed as a potential DNA vaccine
which can provide both humoral and cell-mediated immune
responses.
Pseudomonas aeruginosa causes chronic airway infections in
cystic fibrosis. Recently, abnormally thickened mucus layer
lining the patient airway was proved to be anaerobic. But, how
virulence features of this pathogen are modulated upon
anaerobic growth is largely unknown. Secretion of elastase, a
virulence factor whose production is controlled by LasI-R
quorum sensing, was highly suppressed under the anaerobic
growth condition. qRT-PCR analysis showed that transcription
of elastase-coding lasB gene was completely abrogated in
anaerobically grown PAO1 suggesting that repressed elastase
secretion is due to the regulatory modulation that occurs at the
transcriptional level. These results suggest that a certain
anaerobiosis-induced factor may bind specifically to lasB
promoter to suppress its transcription. Together, our data
suggest that anaerobiosis-induced metabolic shift causes a
modulation of QS machinery in P.aeruginosa.
D030
[This study was supported by Yonsei University College of
Medicine, Intramural Grant Support for Starting Faculty, 62009-0055 to SSY and National Research Foundation of Korea
funded by the Ministry of Education, Science and Technology,
7-2009-0524 to SSY.]
D032
Evaluation of the Immune Response and Protection
Against Botulinum Neurotoxin Type A in ToxoidImmunized Mice
Antibiotic Resistant Characteristics and Identification of Bacteria Isolated from Diarrheic Feces in
Korean Native Beef Calves
Jeong-Hee Kim*, Na-Ri Shin, YunJeong Kim, Gi-eun Rhie,
and Cheon-Kwon Yoo
Division of High-risk Pathogen Research, Center for Infectious
Diseases, National Institute of Health
Yun-Jung Lee*, Jae-Won Park, Sang-Buem Cho, and
Soo-Ki Kim
Department of Animal Sciences and Environment, Konkuk
University
Botulism occurs by the intoxication with botulinum neurotoxin,
resulting in flaccid paralysis. Botulinum neurotoxoid is a major
component which has been classically used for protection
against botulism. In this study, we examined immune
responses against botulinum neurotoxin type A (BoNT/A) in
mouse model. Two mouse strains, BALB/c and C57BL/6,
were immunized three times with BoNT/A toxoid and we have
challenged with BoNT/A. BoNT/A challenge into mice
showed that complete protection against BoNT/A. In order to
know the aspect of immune response in challenged mice, we
measured IgG subclass in sera and, in vitro, spleen cell
cytokine secretion in response to BoNT/A 4 weeks after last
vaccination. In mouse sera, IgG1 antibodies were predominantly developed by BoNT/A vaccination in both strains. In
cytokine assay, IL-4, IL-5, IL-10 and IL-13, Th2-type
cytokines, were detected, and Th1-type cytokines, IL-2 and IL12, were also detected in spleen cell culture supernatants. But
TNF-α and IFN-γ were not detected. These data suggest that
the spleen cells from BoNT/A-vaccinated mice might be a
mixed Th1/Th2 cells although Th2 response is predominant
against BoNT/A toxoid in sera.
Diarrhea in new born calves is serious disease which induces
high mortality, delayed growth and economical loss in farm.
Escherichia coli is an important causative factor in diarrhea.
Bacterial infection and virulence is highly related to quorum
sensing (QS). QS has been regarded as cell-to-cell communication and virulence of pathogenic bacteria. Therefore, understanding of QS is a promising way to prevent disease. In this
study, bacterial strains related to QS were screened from the
fecal specimen of Korean native calves that showed the
symptom of diarrhea. Eleven strains were identified as E. coli
using 16S rRNA gene analysis and their phenotype and
Caenohabditis elegans killing activity were investigated. In
swimming activity, 2201, 2310, 2312 and 2318 showed low
activity and 2205, 2247, 2292, 2298, 2337, 2341 and 2387
were high in activity. In swarming activity, 2201, 2205, 2292,
2298, 2310, 2312 and 2318 were low and 2247, 2337, 2341
and 2387 were high. In protease activity, only 2292, 2337 and
2341 showed activity. In biofilm production, all of isolates
showed similar activity. In C. elegans killing assay, 4 to 9 days
of survival were detected.
176
Poster Sessions
D033
D035
Screening of Burkholderia sp. Diguanylate Cyclase/
Phosphodiesterase Mutant Using Caenorhabditis
elegans Model and Analysis of Its Phenotypes
*
Yun-Jung Kim , Yun-Jung Lee, Sang-Buem Cho, and
Soo-Ki Kim
Department of Animal Sciences and Environment, Konkuk
University
Quorum sensing(QS) controls various mechanisms in bacteria
such as the expression of virulence factor, biofilm formation
and luminescence. Similar to QS, cyclic di GMP also controls
these mechanisms, however its function shows opposite way to
QS and a negative feedback control between QS and c-di-GMP
also has been reported. c-di-GMP is produced by diguanylate
cyclase(GGDEF) and degraded by phosphodiesterase(EAL).
Recently, it has been reported that GGDEF and EAL control
the concentration of c-di-GMP and depending upon its
concentration, bacterial motility, biofilm formation, virulence
and extracellular saccharide production are controlled. Especially,
high virulence activity was found at low concentration of c-diGMP. In this study, virulence and QS of Burkholderia sp. were
investigated by transposon mutagenesis and C. elegans killing
assay. Through the screening of mutants, the strain that showed
lower C.elegans killing activity than wild type was isolated
and the mutagenesis was detected at protein coding region
which produce RNaseII stabilizer including GGDEF and EAL
in Burkholderia sp. 383 chromosome 2. Subsequently, various
phenotypes of isolated strain were investigated.
D034
Proteomic Analysis of Secretory Proteins from the
Streptococcus pneumoniae BAA-255
Chi Won Choi*, Sang Oh Kwon, Sung Ho Yun, and
Seung Il Kim
Division of Life Science, Korea Basic Science Institute
Streptococcus pneumoniae is an important human pathogen
that causes a variety of diseases, such as pneumonia,
bacteremia, meningitis, otitis media, and sinusitis, in both
adults and children. Especially, the morbidity and mortality
rates associated with S. pneumoniae remain very high
worldwide. Because of their clinical importance, genome
sequencing of more than 11 stains were completed and
available. However, proteomic studies of these stains were not
advanced up to now. In the present study, supernatant of
secreted proteins of S. pneumoniae BAA-255 were enriched
with ammonium sulfate precipitation and 1-DE LC-MS/MS
was performed to identify secretory proteins of S. pneumoniae.
48 proteins (21.5% of total identified proteins) were analyzed
to have the signal peptide. 8 cell wall and 6 extracellular
proteins were included in the identified proteins. Among the
identified secretory proteins, PrtA, AmiA, ZmpB, NanA, Gsp781, and GlnP were known virulence factors. Our result
suggests that secretome analysis of S. pneumoniae is useful
approach to identify the virulence factors or biomarkers for
diagnosis and drug targets.
[Supported by grant from Korea Basic Science Institute KMeP]
D036
Screening of a Virulence Gene in Burkholderia sp.
by Using Caenorhabditis elegans Model
Role of National Culture Collection for Pathogens
(NCCP) As a Central Pathogen Bank in Korea
Young-Hee Lee*, Yun-Jung Kim, Yun-Jung Lee,
Sang-Buem Cho, and Soo-Ki Kim
Department of Animal Sciences and Environment, Konkuk
University
Kyenam Lee, Juhee Heo, Kyung-Tae Jung, Hae-Seul
Jeong, Won-Sun You, So-Jung Yoon, Cheon-Kwon Yoo,
and Kwang-Jun Lee
Division of High-risk Pathogen Research, Center for Infectious
Disease, National Institute of Health, KCDC, Korea
Burkholderia sp., a gram negative bacteria, has been regarded
as a pathogenic bacteria causing respiratory disorder in animal
and it is also occasionally causative to human. Especially, the
infection in the patient of cystic fibrosis is critical because it
reduced pulmonary function and increase mortality. Recently,
Caenorhabditis elegans has been used for the research of
bacterial virulence because various pathogenic bacteria or
fungi showed killing activity against C. elegans. In this study,
to construct transposon library, the conjugation between E. coli
that carrying pRL27 and Burkholderia sp. was performed. To
search virulence gene of Burkholderia sp., replica with five
thousands Tn mutants was performed on 96-well plate
containing both of NGM (kanamycin 25 ug/mL, ampicillin 50
ug/mL) and five to ten of C. elegans which is on L4 larval
stage. One hundred six mutants that showed lower killing
activity than wild type were found. Subsequent killing assay
was performed for the verification. Finally, nineteen mutants
that lost virulence were isolated. The phenotypes such as
biofilm formation, swimming/swarming activity and protease
were investigated with isolated mutants.
National Culture Collection for Pathogens (NCCP) is a
national pathogenic resource bank whose function focuses on
the deposition, preservation, development and distribution of
high quality pathogens including high-risk pathogens. NCCP
was established in 1972 by KNIH, joined the WFCC in 2004.
According to the international criteria, NCCP was officially
certified for ISO 9001 in 2009 and strictly managed to become
the global leader. NCCP has established the network, 3 of
specialized pathogen banks in KNIH for pathogens isolated
from infectious diseases patients designated by law, 2 of
collaborative pathogen banks for special pathogens difficult to
collect, and 3 of regional pathogen banks for pathogens
isolated from clinical specimens with clinical information.
NCCP has provided financial support as well as standard
operation protocols for management of unit banks and quality
control of pathogens, and established the network between
central and unit banks. Informational network was constructed
and operated by BIMS. NCCP works hard to provide high
quality services to the whole research community and to
connect with other resource centers to play the role of a central
pathogen bank.
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E001
E003
Characterization of a Novel β-Mannanase from
Paenibacillus sp. HP-8 Isolated from the Gut of
Moechotypa diphysis
Enzymatic Characteristics of a Modular Xylanase
(XylE) from Cellulosimicrobium sp. AG-56 Overproduced in Escherichia coli BL21
Su-Jin Ham1*, Do Young Kim1, Lan Hee Lee1,
Hyun Ju Lee1, Kyung Sook Bae2, Kwang-Hee Son1, and
Ho-Yong Park1
1
Industrial Bio-materials Research Center, Korea Research
Institute of Bioscience and Biotechnology, 2Biological Resources
Center, Korea Research Institute of Bioscience and Biotechnology
Hyun Ju Lee*, Do Young Kim, Su-Jin Ham, Lan Hee Lee,
Kwang-Hee Son, and Ho-Yong Park
Industrial Bio-materials Research Center, Korea Research
Institute of Bioscience and Biotechnology
Paenibacillus sp. HP-8 isolated from the gut of a longicorn
beetle, Moechotypa diphysis, secreted a β-mannanase (ManH)
a molecular mass of approximately 32.0 kDa when cultivated
on a M9 medium containing 0.5% locust bean gum. In contrast
to other well-known mannanases, ManH bound weakly to a
cation exchanger such as CM-Sepharose. The ManH contained
an amino acid sequence of APSFAVGADFSYVPG at its Nterminus, which did not show any high sequence identity with
other similar enzymes. The maximum hydrolysis activity of
ManH toward locust bean gum was observed at pH 7.5 and
55°C where the enzyme retained 50% of its original activity
for 25 min. The enzyme was completely inhibited in the
presence of 1 mM Hg2+ and 5 mM N-bromosuccinimide. In
addition, partial inhibition (>20% of its original activity) of
ManH was observed when incubated with some metal ions
Ni2+, Fe2+, Ca2+, Zn2+, Cu2+, and Sn2+. The specific activity of
ManH toward locust bean gum was determined to be 7,804
IU/mg, indicating that it is a highly active β-mannanase.
E002
The novel modular xylanase (XylE) consisting of an Nterminal catalytic GH10 domain, fibronectin type 3 (Fn3)
domain, and C-terminal carbohydrate-binding module 2 (CBM
2) from Cellulosimicrobium sp. AG-56 that was generated by
the mutation of T440A was biochemically characterized and
the enzymatic properties of XylE were compared to its
processed forms. The maximum catalytic activity of XylE
toward birchwood xylan was observed at pH 6.5 and 40°C.
The enzyme retained >80% of its original activity at the range
of pH 6.0-7.0 when preincubated for 1 h at 4°C. XylE was
thermolabile because the enzyme lost >30% of its activity even
at 35°C after the preincubation of 1 h. The binding affinities of
XylE to various types of insoluble carbohydrate polymers
clearly suggested that Fn3 and/or CBM 2 participated in
substrate-binding because the Fn3-CBM 2-lacking XylE did
not bind to glucose-based polysaccharides.
E004
Characterization of a Novel β-Mannanase from
Cellulosimicrobium sp. AG-56 Isolated from the Gut
of Eisenia fetida
Acid Tolerance Response (ATR) was not Induced in
Leuconostoc mesenteroides ATCC 8293 by PreAdaptation in Mild Acid Condition
Su-Jin Ham1*, Do Young Kim1, Hyun Ju Lee1,
Kyung Sook Bae2, Kwang-Hee Son1, and Ho-Yong Park1
1
Industrial Bio-materials Research Center, Korea Research
Institute of Bioscience and Biotechnology, 2Biological Resources
Center, Korea Research Institute of Bioscience and Biotechnology
Ji Eun Kim* and Nam Soo Han
Department of Food Science and Technology, Research Center
for Bioresource and Health, Chungbuk National University
The gut bacterium of Eisenia fetida, Cellulosimicrobium sp.
AG-56 was able to efficiently degrade mannans as well as
xylan polymers. This organism produced a highly active βmannanase (ManA) with a molecular mass of approximately
37 kDa when cultivated on a M9 medium containing 0.5%
locust bean gum as a sole carbon substrate. The highest
catalytic activity of ManA toward locust bean gum was
measured at 65°C in glycine-NaOH buffer (pH 9.0). At 65°C,
the half-life of the enzyme was evaluated to be approximately
10 min. ManA was completely inactivated in the presence of 1
mM Hg2+ and 5 mM N-bromosuccinimide but not affected
considerably by other divalent cations and chemical reagents
such as Fe2+, Mn2+, Cu2+, Co2+, EDTA, sodium azide, and
Triton X-100. The enzyme could decompose mannosic
polysaccharides such as locust bean gum and guar gum but not
degrade CMC, birchwood xylan, starch, and p-nitrophenolsugar derivatives. The molecular characteristics of the enzyme
(ManA) will be also presented.
178
Poster Sessions
Leuconostoc mesenteroides is commercially important lactic
acid bacterium currently used as a starter culture for kimchi.
However it has been tackled due to the acid sensitivity of this
species. Induced acid tolerance defines a condition whereby,
during exposure to mild acidic conditions, bacteria acquire the
ability to survive lethal acid concentrations. This inducible
mechanism is referred to as the acid tolerance response (ATR)
This study was carried out to investigate the induction effect of
pre-adaptation of L. mesenteroides ATCC 8293 in mild acid
condition (pH 5.0) for 1, 2, and 3h for resistance to normally
lethal intensity of acidic stress (pH 4.0). The survival of acidadapted and non-adapted cells of L. mesenteroides in
phosphate buffer solution (PBS pH 4.0) revealed that acid
adaptation has no influence on the increased tolerance of L.
mesenteroides in PBS (pH 4.0).When compared the initial
CFU/ml, the non-adapted cells and acid adapted cells were
both decreased approximately 100 and 10000 fold subsequently
during the incubation. This result has shown that acid tolerance
is not induced by acid pre-adaptation and instead, pre-acid
exposure may damage L. mesenteroides.
E005
E007
Bacterial Gamma-Glutamyltranspeptidase as a Novel
Bone-Resorbing Factor
The Involvement of Bacteriophytochromes in
Quorum Sensing in Rhodobacter sphaeroides 2.4.1
Jinmoon Kim*, Ho Gil Choi, Minyoung Kim, Eun Jung Bak,
and Jeong-Heon Cha
BK21 Project, Yonsei University, College of Dentistry
Hae-Seon Kim1,2*, Ki-Hoon Yang1,2, Kwang-Eun Jung1,2,
and Jeong-Il Oh1,2
1
Department of Microbiology, 2Pusan National University
γ-Glutamyl Transpeptidase(GGT) was recently identified as a
bone-resorbing factor. GGT is widely distributed in living
organisms, and Bacillus subtilis GGT exhibits high similarity
to the mammalian GGTs. B. subtilis and periodontitis-related
Fusobacterium nucleatum GGTs were examined to determine
if they could induce osteoclastogenesis. Recombinant GGTs
(rGGTs) of B. subtilis and F. nucleatum purified in E. coli
were treated to the co-cultures of osteoblast and bone marrow
macrophages. All rGGTs stimulated osteoclastogenesis and
expression of RANKL. Also, large subunit of B. subtilis rGGT
induced osteoclastogenesis much more than its small subunit.
In addition, osteoclastogenesis induced by rGGT was blocked
by OPG. We present that bacterial GGT is able to induce
osteoclastogenesis via RANKL-dependent pathway and
osteoclastogenic activity of B. subtilis GGT is mostly located
in its large subunit. We suggest that GGT of periodontopathogens can cause bone destruction in periodontitis.
Bacteriophytochrome (Bph) is a red/farred-responsive photoreceptor in bacteria. Bphs were identified to regulate photosystem synthesis in many photosynthetic bacteria. Rhodobacter
sphaeroides 2.4.1, one of the purple non-sulfur photosynthetic
bacteria, has two Bphs (BphG1, BphG2). Unlike most Bphs in
photosynthetic bacteria, BphG1 and BphG2 contain the
GGDEF and EAL domains. They are involved in synthesis and
degradation of c-di-GMP which is a second messenger used in
bacterial signal transduction, especially quorum sensing. The
expression of cerI and cerR encoding the autoinducer synthase
and its transcriptional activator, respectively, in the wild-type
strain of R. sphaeroides was increased in a cell-density
dependent manner and reached the maximum level in stationary
phase. In contrast, their expression in the BphG12 double
mutant was fully derepressed even in the low cell density and
remained constant regardless of the cell density. The secretion
of exopolysaccharide in the BphG12 double mutant was
substantially increased when compared with WT strain. On the
basis of these results, we suggest that Bphs might be involved
in the regulation of quorum sensing in R. sphaeroides.
[This research was supported by Basic Science Research
Program through the National Research foundation of
Korea(NRF) funded by the Ministry of Education, Science and
Technology (R13-2003-013-04002-0).]
E006
E008
Inhibitory Effect of Wogonin on LPS-Induced
Osteoclastogenesis
Tartrate/Succinate Antiporter and Its Regulation in
Escherichia coli
Jinmoon Kim1,2*, Sungil Jang1,2, Eun Jung Bak1,2,
Minyoung Kim1,2, Won-Yoon Chung1,2, Jeong-Heon Cha1,2,
and Yun-Jung Yoo1,2
1
Department of Oral Biology, BK21 Project, Oral Science
Research Center, and Research Center for Orofacial Hard
Tissue Regeneration, Yonsei University College of Dentistry,
2
Department of Applied Life Science, The Graduate School,
Yonsei University
Ok Bin Kim1,2* and Gottfried Unden2
1
Department of Life Science, Ewha Womans University,
2
Institute of Microbiology and Wine Research, University of
Mainz, Germany
To evaluate the inhibitory activity of wogonin against LPSinduced bone resorption, we treated LPS and/or wogonin to the
co-cultures of osteoblasts and pre-osteoclasts and mouse
calvaria, and measured osteoclast differentiation and level of
RANKL, OPG and PGE2. Wogonin inhibited LPS-induced
osteoclastogenesis in co-cultures and LPS-injected mouse
calvaria. In osteoblasts, the up-regulation of RANKL and the
down-regulation of OPG by LPS were inhibited by wogonin.
Wogonin and NS-398, a COX-2 inhibitor, suppressed LPSstimulated PGE2 production in osteoblasts. NS-398 inhibited
the effect of LPS on RANKL and OPG expression in
osteoblasts. These results suggest that wogonin acts as an
inhibitor of LPS induced osteoclastogenesis through downregulation of RANKL and up-regulation of OPG via blockage
of PGE2 production. Thus, wogonin has the potential for use as
a therapeutic agent in bacteria-induced bone resorption.
[This research was supported by Basic Science Research
Program through the National Research foundation of
Korea(NRF) funded by the Ministry of Education, Science and
Technology (R13-2003-013-04002-0).]
E. coli fermentates L-tartrate by the use of L-tartrate/succinate
antiporter TtdT and L-tartrate dehydratase TtdAB. The
transporter TtdT is required for the uptake of L-tartrate. The
growth by L-tartrate and degradation of L-tartrate are
destroyed by the deletion of ttdT. The gene ttdT is located
downstream of the ttdA and ttdB genes, encoding the L-tartrate
dehydratase, and the three genes form an operon ttdABT.
The transport modus of the secondary carrier TtdT and its
relation to the general C4-dicarboxylate carriers were studied.
TtdT catalyze L-tartrate or succinate uptake and specific
heterologous L-tartrate/succinate antiport, preferentially in the
direction of L-tartrate uptake and succinate efflux. The Dcu
carriers do not support L-tartrate transport.
Expression of the ttdABT operon was stimulated by the
LysR-type regulator TtdR in the presence of L- and mesotartrate, and repressed by O2 and nitrate. Expression of ttdR
was repressed by O2, nitrate and glucose, and positively
regulated by TtdR and DcuS. Purified TtdR bound to the ttdRttdA promoter region specifically.
[Supported by grants from DFG and Innovationsstiftung
Rheinland-Pfalz]
179
www.msk.or.kr
E009
E011
Generation of Phlebia tremellosa Transformants
Generating Two Lignin Degrading Enzymes with a
New Expression Vector
Analysis of Pyranose Oxidase Expression Under
Degradation Conditions of Diethylphthalate in
Phlebia tremellosa
Hyun-Woo Kum1*, Sun-Hwa Ryu2, Sung-Suk Lee2, and
Hyoung T Choi1
1
Department of Biochemistry, Kangwon National University,
2
Division of Forest Bioenergy, Korea Forest Research Institute
Baek Joong Kim*, Hyang Soon Lim, Hye Won Kim, and
Hyoung Tae Choi
Molecular Microbiology Lab, Department of Biochemistry,
Kangwon National University
A white rot basidiomycete Phlebia tremellosa secretes laccase
and peroxidase which are involved in the degradation
polymeric lignin and many kinds of endocrine disrupting
chemicals (EDCs). P. tremellosa does not secrete manganese
peroxidase (MnP) in various liquid media which is also a
major component of the lignin degrading enzymes. In order to
get high MnP and Laccase producing transformants of P.
tremellosa, an expression vector (pBARPTLac-TVMnP) has
been constructed using Trametes versicolor MnP cDNA and
Plebia Tremellosa Laccase genomic DNA. Genetic transformation of P. tremellosa was successfully carried out using the
restriction enzyme mediated integration (REMI). Integration of
transforming plasmid has been confirmed using PCR with bargene specific primers and laccase, MnP specific primers. The
expression of the introduced MnP and laccase under
degradation conditions for endocrine disrupting chemicals was
analyzed.
Explosives, insecticides and plasticizers are degraded very
slowly in nature, and show harmful effects by disturbing
endocrine system even at low concentrations. The sex hormone
system is the first target of these chemicals. White rot fungi,
degrading lignin, can degrade various recalcitrant compounds
including the endocrine disrupting chemicals (EDCs). Lignin
peroxidase (Lip) and manganese peroxidase (MnP) are
involved in lignin degradation as well as EDC degradation.
They both require H2O2 for their degrading reactions. Pyranose
oxidase is in charge of suppling H2O2in white rot fungi. We
have isolated a pyranose oxidase cDNA from EDC-degrading
culture of Phlebia tremellosa using the RACE-PCR technique.
In order to find out the relationship between expression of
pyranose oxidase and degradation EDCs by P. tremellosa, the
fungus was grown in a minimal medium with or without
diethylphthalate (DEP), and then the pyranose oxidase
expression was compared by the RT-PCR.
E010
E012
Expression of a Chitinase of Coprinellus congregates
Using Acid Inducible Promoter
Cloning of a Chitinase 2 Gene from Coprinellus
congregatus
Yu Ri Kang* and Hyoung Tae Choi
Molecular Microbiology Lab, Department of Biochemistry,
Kangwon National University
Hye Won Kim*, Sun Uk Bak, and Hyoung Tae Choi
Molecular Microbiology Lab, Department of Biochemistry,
Kangwon National University
Coprinellus congregatus highly expresses chitinase at matured
mushroom stage. When the chitinase cDNA was induced in the
budding yeast, the transformed yeast cells stopped their growth,
and it was almost impossible to get large amounts of chitinase
protein. For chitinase protein production, we have constructed
an acid inducible expression system using the acidic laccase
promoter which was isolated from C. congregatus. Acidic
laccase (lac2) is only expressed under acidic culture conditions,
and we have constructed an expression vector using the acidic
laccase promoter and chitinase cDNA. This construct had been
transformed to C. congregatus in order to get the expression
the chitinase. The successful integration of the expression
vector in the transformants was confirmed by PCR with
vector-specific primers. Two monokaryotic strains having the
constructs were mated to get dikaryons which showed acid
induction of lac2. We expect the expression of the chitinase
protein in the supernatant of acidic liquid medium (pH 4.1).
We will perform the protein purification and its biochemical
characterization.
Fungal cell walls consist of various glucans and chitin. Two
monokaryotic strains of C. congregatus, Cc16 and Cc44, were
mated and cultured in 25°C for 5day to isolate RNA. A
chitinase cDNA from the hyphal cells of C. congregatus was
successfully isolated using the rapid amplification of cDNA
ends (RACE)-PCR technique. The deduced amino acid
sequence of the cDNA has the conserved catalytic domain as in
other fungal chitinase family 18. It showed higher expression
at the matured mushroom stage than the hyphal growth stage,
and the new chitinase cDNA was designated as chitinase 2
gene (chi2). We have also performed cloning of the promoter
region of chitinase 2 from C. congregatus using DNA walking
and RACE-PCR techniques. We have successfully cloned the
full-length chitinase gene involved in the autolysis of
mushroom in C. congregatus. To figure out the function of the
gene, we will construct of knock-out mutant of these gene.
180
Poster Sessions
E013
E015
Functional Expression of Trametes versicolor Cellobiohydrolase and Phlebia tremellosa Laccase in a
Brewing Yeast
Novel Deblocking Aminopeptidases of Hyperthermophilic Archaeon Thermococcus onnurineus NA1
Sun Jue Park , Hye Young Kim, and Hyoung Tae Choi
Molecular Microbiology Lab, Department of Biochemistry,
Kangwon National University
Yeol Gyun Lee1*, Sung Gyun Kang2, Jung-Hyun Lee2,
Jong-Soon Choi1, Seung Il Kim1, and Young-Ho Chung1
1
Division of Life Science, Korea Basic Science Institute, 2Marine
Biotechnology Center, Korea Ocean Research & Development
Institute
White rot basidiomycetes Phlebia tremellosa and Trametes
versicolor have enzyme system for the degradations of
polymeric lignin, cellulose and hemicellulose. In T. versicolor,
the cellobiohydrolase (Cbh) separates one cellulose chain at a
time from cellulose and hydrolyses it, then Cbh releases
cellobiose from the ends of cellulose molecules. In order to get
high cellobiohydrolase and laccase producing transformants of
yeast, two expression vectors (YIPGTVcbh, YIPGPTlac) have
constructed using T. versicolor cellobiohydrolase cDNA and P.
trmellosa laccase cDNA. We have used glyceraldehyde-3phosphate dehydrogenase promoter (GPD promoter) to get
constitutive expression. We have transformed the expression
vectors to yeast in order to get transformants having increased
degrading activities of lignin and cellulose. Transformation of
yeast was successfully carried out using two expression vectors
(YIPGTVcbh, YIPGPTlac), and we have confirmed an activity
and an expression of two enzymes of yeast transfomants. We
will perform one-step ethanol production from acid
hydrolysate of wood using the transformants.
It has been reported that one of the hyperthermostable
deblocking aminopeptidase (DAP) from Thermococcus
onnurineus NA1 (TNA1_DAP) exhibits hydrolytic activity
toward short peptides and acyl-peptides. In the genome
database of T. onnurineus NA1, three new open reading frames
homologous to the DAP of T.onnurineus NA1 were found.
The deduced amino acid sequences were similar to DAPs of
pyrococcus furiosus and pyrococcus horikoshii, a member of
peptidase family M42.The three new genes for the proteins
were cloned and overexpressed in Escherichia coli. Molecular
masses assessed by SDS-PAGE were 38kDa, 37kDa and
36kDa respectively, and exhibited aminopeptidase activity,
including deblocking activity. One of the purified DAPs
showed the optimum activity at 90°C and its half-life (t1/2) was
2.5 hr. It was confirmed that T. onnurineus NA1 has four
similar aminopeptidases with deblocking activity and that these
enzymes appear to play important roles in hydrolyzing small
and acyl-peptides in T.onnurineus cells.
*
[Supported by the Marine and Extreme Genome Research
Center Program of the Ministry of Land, Transport and
Maritime Affairs]
E016
E014
Medium Optimization for the Production of
Nattokinase by Bacillus subtilis NK7-1 Using
Response Surface Methodology
1*
2
1
Hee-Jong Yang , Moon Chang Kim , Nack-Shick Choi ,
Seong-Yoep Jeong1, Keug-Hyun Ahn1, Chan-Sun Park1,
Yong-Il Hwang2, Byung-Dae Yoon1, and Min-Soo Kim1
1
Institute Bioindustry Research Center, Korea Research Institute
of Bioscience and Biotechnology, 2Department of Food Science
and Biotechnology, Kyungnam University
Bacillus subtilis NK7-1 producing a potent fibrinolytic enzyme
was isolated from Chungkook-Jang a traditional fermented
food in Korea. Nattokinase is a potent fibrinolytic enzyme that
is considered to be a promising agent with the potential for
fighting cardiovascular diseases. Response surface methodology
(RSM) and statistical analysis system (SAS) 9.1 program were
employed to optimize a fermentation medium for the
production of nattokinase by B. subtilis NK7-1. The three
variables involved in this study were beef extraction, peptone,
and initial pH. Using this methodology, the optimum values of
the critical components for maximum production of
nattokinase for concentration of beef extraction, peptone, and
initial pH were 0.4%, 0.28%, and 8.84, respectively. Analysis
of variance (ANOVA) showed a high coefficient of determination (R2) value of 0.9717. Using the optimized condition,
nattokinase production by B. subtilis NK7-1 was obtained at 24
h-cultivation.
Inhibition of Bacillus cereus by Bacteriocin from
Lactococcus lactis ET-45 in Fermented Kimchi
Seong-Yoep Jeong1,2*, Chan-Sun Park1, Nack-Shick Choi1,
Hee-Jong Yang1, Da-i Jung1, Keug-Hyun Ahn1,
Dae-Ook Kang2, Byoung-Dae Yoon1, and Min-Soo Kim1
1
Jeonbuk Branch Institute Bioindustry Research Center, Korea
Research Institute of Bioscience and Biotechnology, 2Department
of Biochemistry and Health Science, Changwon National
University
Bacteriocin producting lactic acid bacteria were isolated from
Kimchi. Strain ET-45 was identified as L. lactis subsp. lactis
based on sugar fermentation pattern test using API 50 CHL.
The 16S rDNA sequence of strain ET-45 showed 99% identity
to those of reference strain of L. lactis. The bacteriocin
exhibited inhibitory activity against Bacillus cereus, Leuconostoc mesenterides, Leuconostoc carnosum, Leuconostoc
lactis, Leuconostoc mesenteroides subsp. Cremoris, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus
viridesceus, Pediococcus dextrinicus, and Enterococcus
faceium. Optimal production of the bacteriocin was at pH 7.5
and 30°C for 18 ~24 h. Sucrose and protease peptone are
essential for bacteriocin production as carbon source and
nitrogen source, respectively. Antibacterial activity of the
bacteriocin was completely disappeared by proteinase K,
which indicates it is proteinous nature. The bacteriocin was
fully stable at 121°C for 60 min. Solvents such as chloroform,
ethanol, acetone, acetonitrile, hexane, isopropanal did not
effect on the activity. The molecular weight of bacteriocin was
estimated to be about 1.3 ~ 2.0 kDa by Tricine-SDS-PAGE.
181
www.msk.or.kr
E017
E019
Biological Activity of Extracts from Hypholoma
sublateritium
Insights into SUMO Function under Oxidative
Stress in Saccharomyces cerevisiae
Sunga Choi1*, Jung-A song1, Donghyun Kim1,
Gilhwa Jeong2, Jihong Kim3, and Jongwoon Choi2
1
Department of Life Science, Hallym University, 2Forest
Research Institute of Gangwon-do, 3Department of Forest
Management, Kangwon National University
Gyu-bum Lim* and Won-Ki Huh
School of Biological Sciences, and Research Center for Functional
Cellulomics, Institute of Microbiology, Seoul National University
This study was first carried out to investigate the biological
activities of Naerypholoma sublateritiumi. The major chemical
components of N. sublateritiumi were analyzed. There were
not large differences in chemical contents of carbohydrates,
fatty acid and ash content: however, total protein contents were
19.3%, relatively higher. As a result of comparing with antilipid peroxidation of each extract, the effects were highest in
hexane and dichloromethan extracts. For analysis of antiproliferation activity, we used five cancer cell lines, MDA-MB
231 and MCF7 (Breast cancer cells), HT29 (Colon cancer cell),
HeLa (cervical cancer cell) and SKOV 3 (ovarian cancer cell).
Dichloromethan and ethyl acetate extracts showed an effective
decrease of cancer cells viability in time- and concentrationdependent manner. These results of the present study show that
the ability of N. sublateritiumi extracts might be a promising
agent for performing clinically useful chemotherapy against
diverse ailments as well as cancer.
[This paper consisted by assistances of a technology development assignment scientific forest(과제번호: S120909L110120).]
Since the discovery of ubiquitin in the mid-1970s, an entire
family of small proteins related to ubiquitin has been defined.
Among them, SUMO (small ubiquitin-like modifier) is highly
conserved in eukaryotic cells and a budding yeast Saccharomyces cerevisiae has a single SUMO protein which has also
been termed Smt3. Although the function of Smt3 has not been
defined clearly, it is expected to control the activity or
localization of target proteins under various environmental
conditions. To examine this possibility, we performed a
genome-wide analysis of in vivo protein sumoylation in S.
cerevisiae using the bimolecular fluorescence complementation (BiFC) assay under oxidative stress. 5,911 yeast strains
expressing C-terminally VN-tagged proteins (representing
~95% of the yeast proteome) were mated with a strain
expressing N-terminally VC-tagged Smt3 and all the resulting
diploid cells were analyzed by the BiFC assay in the presence
or absence of hydrogen peroxide. Through this analysis, we
identified several proteins showing interesting changes in the
BiFC signal pattern. Based on the obtained results, we suggest
a potential role of SUMO under oxidative stress.
E020
E018
Expression of amyX and ytlR Genes that Play
Important Role in Glycogen Metabolism of B.
subtilis
*
Tianshi Wang , Jung-Hoon Choi, and Jung-Wan Kim
Department of Biology, University of Incheon
B. subtilis, an endospore forming bacterium, undergoes
alternative life cycle by environmental cues. Pullulanase plays
important role in glycogen accumulation, which is likely to be
closely related to determination of cell’s destiny. Its gene is
located in a gene cluster of ytlP-ytlQ-ytlR-amyX-ytmP with no
known function. YtlR shares 50% homology with a transcriptional regulator of Lactobacillus reuteri, suggesting that it
might be a novel regulator. The ytlR mutation caused decrease
of sporulation efficiency and increase of glycogen accumulation. The expression of the genes was monitored using PamyX or
PytlR-lacZ fusion in B. subtilis. Both promoters were induced
mostly at the beginning of stationary phase and reached the
highest level during endospore formation in both 2XSG and
defined media. The promoters were induced earlier and higher
levels in the presence of β-cyclodextrin (CD), fructose, or
sucrose. The promoters were expressed at low level in the
presence of maltodextrin or glycerol and at the highest level in
the presence of β-CD. The results imply that the two genes are
expressed coordinately and might be under the control of
various regulators.
Characteristic of PR Homologues from Marine
Bacterioplanktonic SAR116 and SAR11 Clades
Ah Reum Choi1*, Hyun-Myung Oh2, Ki Young Lee2,
Jang-Cheon Cho2, and Kwang-Hwan Jung1
1
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University, 2Division of Biology
and Ocean Sciences, Inha University
Proteorhodopsins are light-dependent proton pumps. Proteorhodopsin (PR), retinal-containing integral membrane
proteins, was originally detected in the uncultured marine γproteobacterial SAR86 group. Recently, PRs are discovered
diverse ocean sea water, such as Monterey Bay, Hawaii Ocean,
Palmer station in South Pole, Mediterranean Sea, Red Sea,
Sargasso Sea, and Pacific Ocean. Here, from Western Pacific
Ocean Margin a bacterioplankton designated IMCC1322 was
successfully grow in tubes, it was identified as a member of
uncultured SAR116 clade of α-proteobacterial lineages and
two bacteria from SAR11 group. From SAR116 and SAR11,
we isolated the PRs by arbitary primed PCR. The PR genes
encode a protein of 261 a. a., 262 a. a, and 232 a. a,
respectively. The genes were functionally expressed in E. coli
and bound all-trans retinal to form a pigment (λmax=519 nm,
557 nm, 535 nm at pH 7). In PR of SAR116, titration of Asp105
occurred with a pKa of 5.6. In first PR of SAR11 (9062), one
of Asp102 occurred with a pKa of 9.3. It showed a light-driven
proton pumping activity and the rates of those photocycle are
also fast similar to GPR.
[Supported by grant from KOSEF R01-2008-000-20731-0]
182
Poster Sessions
E021
E023
A Comprehensive In Vivo Analysis of Ras Interactome in Saccharomyces cerevisiae Using Bimolecular Fluorescence Complementation Assay
Photoregulation of ASR (Anabaena Sensory Rhodopsin) Through ASRT (Anabaena Sensory Rhodopsin Transducer)
Dae-Gwan Yi*, Eun-Bin Yang, and Won-Ki Huh
School of Biological Sciences, and Research Center for Functional
Cellulomics, Institute of Microbiology, Seoul National University
So Young Kim* and Kwang-Hwan Jung
Department of Life science and Interdisciplinary Program of
integrated Biotechnology, Sogang University
Activation of diverse cell-surface receptors can stimulate the
convergent signals that lead to the activation of Ras. To
identify novel Ras effectors, we performed a genome-wide in
vivo analysis of Ras interactome in Saccharomyces cerevisiae
using the bimolecular fluorescence complementation (BiFC)
assay. 5,911 yeast strains expressing C-terminally VN-tagged
proteins (~95% of the yeast proteome) were constructed and
mated with a strain expressing N-terminally VC-tagged Ras2,
the major Ras protein in S. cerevisiae. Through analysis of all
the resulting diploid cells with the BiFC assay, we identified
several novel candidates showing positive interaction signals
with Ras2. The proteins interacting with Ras2 were found to be
involved in several biological processes, including transport,
lipid metabolic process, and RNA metabolic process. We also
acquired the interactome of constitutively active or inactive
mutant form of Ras2. We hope the analysis of these putative
Ras interactors will provide deeper understanding of the Rasmediated signaling pathways and their cellular functions.
Anabaena Sensory rhodopsin (ASR) from the fresh-water
cyanobacterium Anabaena sp. PCC7120 has a sensory
function and it is believed to play a role as a photoreceptor for
phycobilin expression. Its function appears to involve
modulation of a soluble cytoplasmic transducer (ASRT).
ASRT is a soluble protein and transmitted a light signal to
cytoplasmic part through physical interaction with ASR. We
confirmed that ASR interacts with ASRT through in vitro
binding assay. ASRT was also bound to a promoter region of
cpc, pec, kai ABC and asr operon in gel shift assay and CHIP
assay. It seems that ASRT regulates the photochemical
property of ASR. In this study, we measured the photoconversion rate of ASR without ASRT and with tetramer and
monomer formed ASRT. In the presence of ASRT, the rate is
faster than in the absence of ASRT. And we are trying to
understand the binding affinity between ASR and ASRT or
ASRT and promoter region with each monomer and tetramer
of ASRT protein. We confirmed the binding affinity of ASR
and promoter region with in vivo beta-galactosidase assay and
ITC (Isothermal Calorimetry).
[Supported by 21C Frontier Microbial Genomics and
Application Center Program]
E022
E024
Glycogen Accumulation and Characterization of
Debranching Enzyme in Vibrio vulnificus
1*
1
2
Ah-Reum Han , Yeon Ju Lee , Jong-Tae Park , and
Jung-Wan Kim1
1
Department of Biology, University of Incheon,
2
Department of Food Science, Iowa State University, USA
V. vulnificus is a gram-negative halophilic marine pathogen,
experiencing alternative life cycle between marine environment and bodies of host. Glycogen is major energy source for
Vibrio and other microorganisms. It is also related to
pathogenicity. V.vulnificus accumulated glycogen 10X more
than B.subtilis and E.coli when 1% maltodextrin was supplemented to the media. Glycogen accumulation was dependent
on media and carbon source. Interestingly, glycogen
synthesized by V.vulnificus had shorter side chains compared
to glycogen from B. subtilis and E. coli. Analysis of V.vulnificus
genome revealed that constitution of genes for glycogen
metabolism is unique, with two glycogen phosphorylase genes
and three putative debranching enzyme genes. One of
debranching enzyme genes, VV2_1226, that have been
annotated as type II secretory pathway protein of 661 amino
acids with a predicted molecular mass of 76,560 daltons was
cloned on pET28a and over-expressed in E. coli BL21 with
IPTG induction. The protein purified using Ni-NTA column
hydrolyzed glycogen most efficiently but pullulan and
amylopectin only poorly. The optimal pH and temperature for
the enzyme was 6.5 and 35°C, respectively.
The Existence of FD Blh Dioxygenase
You Sun Kim1*, Soon-Kyeong Kwon2,3, Jihyun F. Kim2,4,
and Kwang-Hwan Jung5
1
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University, 2Korea Research
Institute of Bioscience and Biotechnology, 3University of Science
and Technology, 4Korea University of Science and Technology,
5
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University
The microbial rhodopsins are typically seven transmembrane
proteins that use a retinal as a chromophore attached to
conserved lysine residue. A variety of rhodopsins was
discovered in many habitats. Proteorhodopsin (PR) functions
as a light-driven proton pump, which was found in uncultured
marine bacteria of SAR86. We obtained two flavobacterial
strains called IMCC1997 and Donghaeana dokdonensis from
the surface of sea water in Korea East Sea. Interestingly,
IMCC1997 has one PR and D. dokdonensis two types of
microbial rhodopsins that co-exist in the genome. There is a
gene which encodes Blh protein (bacteriorhodopsin-related
protein-like homolog protein) in D. dokdonensis that has been
proposed to catalyze or regulate conversion of β-carotene to
retinal to make rhodopsins in prokaryotes and is located next to
the PR gene but is transcribed differently in Dokdonia sp.
MED134 and Polaribacter sp. MED 152. DD blh showed
enhanced activity compared to PR blh since we could detect
the formation of rhodopsin as pink color even though its
activity is a little bit lower than that mouse dioxygenase.
[Supported by KOSEF R01-2008-000-20731-0]
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E025
E027
Expression and Characterization of ARⅡ(Acetabularia RhodopsinⅡ)
Comparative Study of Microbial Rhopdopsins
Between Arctic and Antarctic Ocean
Se Jun Kim* and Kwang-Hwan Jung
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University
Byung Hoon Jung*, Jae Young Jung, and
Kwang-Hwan Jung
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University
The microbial rhodopsins are typically seven transmembrane
proteins that use retinal as a chromophore absorbs light energy
for ion transport or photoreceptor functions. Acetabularia
acetabulum is green algae, which has photosynthetic activity.
The life cycle of A. acetabulum has distinct 3 developmental
stages: juvenile, adult, and reproductive. Juvenile and adult
stages are temporally sequential, but physiologically and
morphologically distinct. It has been discovered bacteriorhodopsin (BR)-like protein and that was called Acetabularia
rhodopsin I (ARI). ARI is intronless and encodes a protein of
246 amino acids with molecular weight of 27 kDa, which
function was characterized to be a proton-pump stimulated by
green light. Recently, it was revealed that new rhodopsin-like
proteins exist in genomes of A.acetabulum which is called new
rhodopsin Acetabularia rhodopsin II (ARII). We expressed the
ARII-mistic fusion in E. coli and purified only ARII by NiNTA affinity chromatography. We measured an absorption
spectra (λmax=532nm) and will study further about its pKa,
photocycle, and pumping activity.
Proteorhodopsin (PR) is discovered from marine bacteria and
is 7-transmembrane light-harvest protein that has all-trans
retinal as a chromophore. PR has a proton pumping activity
from inside to outside of the cell using light energy. Generally,
PR is classified into two groups by the maximum absorption
wavelength. One is the BPR which absorbs blue around 500
nm wavelength and the other is the GPR which absorbs green
(530 nm) region of visible spectrum. Previously, we have
isolated and characterized 18 GPR homologs from the sea near
Svalbard, Norway. We have confirmed the positive-correlation
between proton pumping function and photochemical reaction
rate. In this study, we isolated the conserved regions of
rhodopsin by PCR from the sea near King George Island and
compared with Arctic microbial rhodopsins. We also isolated a
single full sequence of Antarctic rhodopsin and several piece
of conserved regions between helix C and F. We are now
characterizing Antarctic rhodopsin and also trying to express in
E. coli to characterize the photochemical properties.
[Supported by KOSEF R01-2008-000-20731-0]
E026
[Supported by KOSEF R01-2008-000-20731-0]
E028
Purification Method for Donghaeana dokdonensis
Rhodopsin I
Photochemistry of Acetabularia RhodopsinⅠand of
its Important Mutants
Sehwan Kim1*, Soon-Kyeong Kwon2,3, Jihyun F. Kim3,4,
and Kwang-Hwan Jung1
1
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University, 2Korea Research
Institute of Bioscience and Biotechnology, 3Korea University of
Science and Technology, 4Korea Research Institute of Bioscience
and Biotechnology
Keon Ah Lee* and Kwang-Hwan Jung
Department of Life Science and Interdisciplinary Program of
Integrated Biotechnology, Sogang University
Rhodopsins are a family of membrane-embedded photoactive
retinylidene proteins. Rhodopsins have several functions as an
ion pump, sensory receptor and ion channel. Donghaeana
dokdonensis Rhodopsin I (DDRI) is the one of rhodopsin that
is isolated from the Korea East Sea. It has a typical proton
acceptor and donor which mediate proton translocation from
intracellular side to extracellular region when it absorbs light
energy, and its in vitro proton pumping activity has been
demonstrated. It has biophysical features which are very
similar to proteorhodopsin (PR). In this study, we focused on
how to get high yield of pure DDRI. We transformed DDRI to
E. coli UT5600 cell for rhodopsin expression. DDRI expressed
cells lyses by sonicator and proteins are solubilized with
dodecyl maltoside. Since DDRI has six-histidine at c-terminal,
we use Ni-NTA resin for the purification. We tried to remove
non-specific binding with pre-treatment of imidazole when its
binding and repeated twice of Ni-NTA purification. It seems
that the purity is suitable to get a crystal of DDRI.
[Supported by 21C Frontier Microbial Genomics and
Application Center Program]
184
Poster Sessions
Microbial rhodopsins are retinal-binding proteins known as ion
pumps and photosensory receptors. Acetabularia rhodopsin I
(ARI) is from Acetabularia acetabulum, a giant unicellular
green alga (kindly provided by Professor Dina Mandoli). ARI
fused with MISTIC was expressed in E. coli UT5600. Several
properties of ARI were measured; the absorption spectra, pKa
of the acceptor residue, the amount of protons pumped outward,
light-induced difference spectra, and half-life of several
intermediates. Unmisticated ARI showed pink color and its
absorption maximum was around 516 nm. The pKa of ARI
was measured and tested the proton pumping outward activity
at pH 7.5. Two of photointermediates of ARI have been
detected including K (or L)-like and M-like that have absorption maximum at 584 and 396 nm, respectively. We could also
detect the mRNA of Acetabularia rhodopsin in the A.
acetabulum whole cell by RT-PCR. We also constructed 4
mutants, D89N, D89E, D100N, and D100E (D89 and D100
are expected to be acceptor and donor residues, respectively)
mutants and spectroscopic properties of these were also
measured.
[Supported by the 21C Frontier Microbial Genomics and
Application Program]
E029
E031
Transcriptional Regulation of Aldehyde Reductase
YqhD of E. coli by YqhC, an AraC-Type Regulator,
Involving Its 5’-UTR Region
*
Junghoon Lee and Chankyu Park
Korea Advanced Institute of Science and Technology
YqhD is an aldehyde reductase detoxifying glyoxal (GO), a
reactive α-oxoaldehyde accumulated by a stress associated
with glucose metabolism. YqhD was previously characterized
as an enzyme, while its transcriptional regulation mechanism is
unknown. Glyoxal-resistant mutants isolated from yqhC gene,
over-expresses YqhD protein, implying YqhC functioning as a
transcriptional regulator for YqhD. We observed that the level
of YqhD transcript was dramatically increased in the glyoxalresistant mutant. By screening for mutants down-regulating
YqhD, we obtained point mutations in the yqhD promoter and
5’-UTR region. In order to test whether these mutants alter
transcriptional regulation, we carried out a real-time PCR
experiment. The promoter and 5’-UTR mutations lowered the
level of yqhD transcript, suggesting that the YqhC protein and
the RNA secondary structure in 5’-UTR affect YqhD transcription. Furthermore, gel-shift assay revealed that the YqhC
protein binds to the yqhD promoter region that contains AraCbinding consensus sequence.
[Supported by the 21C Frontier Microbial Genomics and
Application Center Program.]
E030
Rapamycin-Induced Abundance Changes in the
Proteome of Budding Yeast
Chun-Shik Shik1*, Yeon-Ji Chang1, Hun-Goo Lee2, and
Won-Ki Huh1
1
School of Biological Sciences, Seoul National University,
2
Department of Molecular Biology and Biochemistry, Rutgers
University
The target of rapamycin (TOR) signaling pathway conserved
from yeast to human plays critical roles in regulation of eukaryotic cell growth. However, due to the functional diversity of
TOR pathway, we do not know yet some key effectors of the
pathway. To find unknown effectors of TOR signaling
pathway, we took advantage of a green fluorescent protein
(GFP)-tagged collection of budding yeast Saccharomyces
cerevisiae. We analyzed protein abundance changes by
measuring the GFP fluorescence intensity of 4156 GFP-tagged
yeast strains under inhibition of TOR pathway. Our proteomic
analysis argues that 83 proteins are decreased whereas 32
proteins are increased by treatment of rapamycin, a specific
inhibitor of TOR complex 1 (TORC1). We suggest that the
115 proteins indentified in this study may be directly or
indirectly involved in TOR signaling and can serve as
candidates for further investigation of the effectors of
TOR pathway.
[Supported by grants from the 21C Frontier Functional
Proteomics Project and the 21C Frontier Microbial Genomics
and Application Center Program.]
E032
Dephosphorylated NPr of The Nitrogen-Metabolic
PTS Regulates Lipid A Biosynthesis by Direct
Interaction with LpxD
1*
1
1,2
Hyun-jin Kim , Chang-Ro Lee , and Yeong-Jae Seok
Department of Biological Sciences and Institute of Microbiology,
Seoul National University, 2Department of Biophysics and
Chemical Biology, Seoul National University
1
Bacterial phosphoenolpyruvate-dependent phosphotransferase
systems (PTS) play multiple roles in addition to sugar transport.
Recent studies revealed that enzyme IIANtr of the nitrogenmetabolic PTS regulates the intracellular concentration of K+
by direct interaction with TrkA and KdpD. In this study, we
show that NPr of the nitrogen-metabolic PTS directly interacts
with and regulates Escherichia coli LpxD which catalyzes
biosynthesis of lipid A of the lipopolysaccharide (LPS) layer
and therefore is essential for growth. LpxD showed a
preferential interaction with unphosphorylated NPr. Mutations
in lipid A biosynthetic genes such as lpxD are known to confer
hypersensitivity to hydrophobic antibiotics such as rifampin; in
contrast, a ptsO (encoding NPr) deletion mutant showed
increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data show that unphosphorylated NPr
decreases LPS biosynthesis by inhibiting LpxD activity.
[Supported by grants from the 21C Frontier Microbial
Genomics and Applications Center Program, Ministry of
Science & Technology (Grant MG08-0201-1-0)]
Quantitative Proteomic Analysis of Ribosomal
Protein L35b Mutant of Saccharomyces cerevisiae
Yong Bhum Song1*, Min A Jhun2, Taesung Park3, and
Won-Ki Huh1
1
School of Biological Sciences, and Research Center for
Functional Cellulomics, Institute of Microbiology, Seoul
National University, 2Bioinformatics Program, Seoul National
University, 3Department of Statistics, Seoul National University
Recent studies have revealed that in higher eukaryotes, several
ribosomal proteins are involved in some pathological events or
developmental defects, indicating that ribosomal proteins
perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal
proteins, we aimed to analyze the changes in proteome
expression in ribosomal protein mutants by using S. cerevisiae
as a model system. We introduced the rpl35bΔ mutation into
the 4,159 green fluorescent protein (GFP)-tagged yeast strains
by using the synthetic genetic array (SGA) method, and
performed quantitative proteomic analysis by using a
multilabel microplate reader and flow cytometer. We identified
22 upregulated and 20 downregulated proteins in the rpl35bΔ
mutant. These proteins were primarily classified into the Gene
Ontology (GO) categories of cellular biosynthetic process,
translation, protein or nucleotide metabolic process, cell wall
organization and biogenesis, and hyperosmotic response. Our
results show that a ribosomal protein mutation can lead to
perturbation in the expression of several proteins, including
some other ribosomal proteins.
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E033
E035
In Escherichia coli, the Regulation of RppH in
Peptidoglycan Synthesis Pathway
Fractionation of Components for Atopy Treatment
from Fermented Products of Herbs
Miri Kim1*, Seung-Hyon Cho1, Hyun-Jin Kim1,
Chang-Ro Lee1, and Yeong-Jae Seok1,2
1
Laboratory of Macromolecular Interactions, Department of
Biological Sciences and Institute of Microbiology, Seoul
National University, 2Department of Biophysics and Chemical
Biology, Seoul National University
Hye Young Kim1*, Sun Wook Park1, Doo Il Rung1,
Sung Gu Lee2, and Hyoung Tae Choi1
1
Department of Biochemistry, Kangwon National University,
2
Mushville
Regulation of cellular mRNAs level is important for metabolite
and homeostasis in the cell to regulate protein level in various
environmental conditions. In prokaryotes, though 5’ end of
mRNA is not modified, it retains the 5’ triphosphate to
stabilize, likely to the cap structure in eukaryotes. RppH
(NudH/YgdP) is a prokaryotic functional homology of a
decapping enzyme in eukaryotic cells to remove 5’
triphosphate to generate a 5’ monophosphate mRNA which is
a substrate for the endocuclease RNase E. Moreover, RppH,
belonging to the Nudix superfamily of enzymes which
hydrolyze Nucleoside diphosphates linked to other moiety X,
is well known for hydolysis diadenosine oligophosphates
(alarmone) in vitro. It was reported that RppH of hydrolyse
activity regulates endogenous alarmone levels in stress. Here
we show that RppH strongly interacts with DapF, diaminopimelate epimerase, which converts LL-DAP into meso-DAP.
Meso-DAP(diaminopimelate) plays a important role as a
precursor in lysine synthesis pathway and peptidoglycan
synthesis pathway. Thus, the function of NudH regulating
dapF activity is meaningful to study in addition to that of
NudH pyrophosphohydrolase and hydrolase activity
E034
Potassium Mediates Escherichia coli Enzyme IIA
Ntr
-dependent Regulation of Sigma Factor
Selectivity
Chang-Ro Lee1*, Seung-Hyon Cho1, Hyun-Jin Kim1,
Miri Kim1, Alan Peterkofsky2, and Yeong-Jae Seok1,3
1
Laboratory of Macromolecular Interactions, Department of
Biological Sciences and Institute of Microbiology, Seoul
National University, 2Laboratory of Cell Biology, National
Heart, Lung, and Blood Institute, National Institutes of Health,
USA, 3Department of Biophysics and Chemical Biology, Seoul
National University
An Escherichia coli mutant devoid of enzyme IIANtr (EIIANtr)
of the nitrogen PTS is extremely sensitive to leucinecontaining peptides due to decreased expression of
acetohydroxy acid synthase. We report here that the leucine
hypersensitivity of a ptsN (encoding EIIANtr) mutant was
suppressed by deleting the rpoS gene, encoding the stationary
phase σfactor. Despite intracellular levels of sigma factors
comparable to the wild-type strain, most of the genes downregulated in a ptsN mutant are controlled by σ70, while all the
up-regulated genes are controlled by σS, implying that the
balance of sigma activities is modified by ptsN deletion. This
change of sigma factor activity was found to be due to
increased levels of K+. In vitro transcription assays showed that
a σ70 controlled gene and a σS controlled gene were differentially affected by potassium concentration. Biochemical studies
revealed that K+ is responsible for sigma factor competition by
differentially influencing the binding of σ70 and σS to core
RNA polymerase. Taken together, the data indicate that
EIIANtr controls sigma factor selectivity by regulating the
intracellular K+ level.
186
Poster Sessions
Atopy is a very notorious disease because there are so many
reasons for the disease, and that is the main reason for
extremely hard to get healing. Since even many foods which
are used in our normal life can cause the disease in young
children, it is highly required to have good treatments with no
side effects. Extracts of four different herbs and cell extract of
Phellinus linteus were fermented with lactic acid bacteria and
budding yeast for 2 months, and the fermented product showed
positive healing effect against atopy when examined at the cell
level experiment. In order to isolate the effective components,
ultrafiltration with stepwise molecular weight cut-off filtration,
and thin layer chromatography have been carried out. Here we
present the possible protocols for the isolation of the effective
chemicals from the fermented mixtures. We have isolated
several chemical mixtures which show good effects, and these
will be isolated into each single chemical to determine their
structure.
F003
F001
Molecular Characterization of FinR, a Novel RedoxSensing Transcriptional Regulator in Pseudomonas
putida KT2440
*
Jinki Yeom and Woojun Park
Division of Environmental Science and Ecological Engineering,
Korea University
FinR is required for the induction of the fpr (ferredoxinNADP+ reductase) under superoxide stress conditions in P.
putida. Many proteobacteria harbor FinR homologues in their
genome as a putative LysR-type protein. When these
conserved cysteines along with two other cysteine residues
present in FinR were individually mutated to serines, the FinR
remained active, unlike SoxR and OyxR in E. coli. The results
of our in vitro DNA-binding assay with cellular extracts
showed that FinR binds directly to the fpr promoter region. In
order to identify the FinR functional domain for sensing
superoxide stress, we employed random and site-directed
mutagenesis of FinR. Interestingly, two mutants (L215P,
D51A) appeared to be constitutively active, regardless of
superoxide stress conditions. Ferrous iron depletion, ferric iron
addition, and fdxA (ferredoxin) gene deletion also participate in
the regulation of fpr. These data indicate that the FinR has
unusual residues for redox sensing and that the redox-sensing
mechanism of FinR differs from the well-known mechanisms
of OxyR and SoxR.
Cell-Free Culture Fluid-Mediated Expression in
Corynebacterium glutamicum
Hee-Sung Shin*, Yong-Jae Kim, In-Hwa Yoo, and
Un-Hwan Ha
Department of Biotechnology and Bioinformatics, Korea
University
Autoinducing expression is an environmental sensing system
that allows bacteria to monitor their own population density. It
is mediated by a diverse signaling molecule that is produced,
released and detected by bacterium itself. However, there have
been no reports on autoinduction-mediated expression in
Corynebacterium glutamicum, an important gram-positive
bacterium widely used for glutamine production. In this study,
we report the identification of genetic loci, including
acyltransferase, whose expression is under the control of cellfree culture fluid from C. glutamicum. It indicates that C.
glutamicum possesses an autoinduction system, producing
signaling molecules to the threshold stimulatory level, resulting
in the induction of acyltransferase identified in this study.
[Supported by grants from CJ Cheiljedang Corporation as a
project of 21C Frontier Microbial Genomics and Applications
Center.]
[This work was supported by a grant from the NCRC (grant #:
20090091491) and KSEF (R01-2008-000-10697-0) program.]
F002
NtrC-Sensed Nitrogen Availability Is Important for
Oxidative Stress Defense in Pseudomonas putida
KT2440
Jinki Yeom* and Woojun Park
Division of Environmental Science and Ecological Engineering,
Korea University
The zwf gene is repressed by NtrC under nitrogen-limited
condition. Previously, we demonstrated that induction of zwf-1
is required for protecting P. putida cells under oxidative stress,
which could be possible probably because of derepression of
HexR on the zwf-1 gene under oxidative stress. These findings
led us investigate that NtrC still represses the zwf-1 under
nitrogen-limited oxidative stress condition, which makes cells
more sensitive under such condition. Interestingly, deletion of
the ntrC gene significantly reduces growth rate, but renders
cells more resistant to oxidative stress, under nitrogen limited
condition in P. putida. The results of transcriptome analysis
demonstrated that the derepression of several oxidative stress
genes along with the zwf-1 gene might confer high resistance
to oxidative stress in the ntrC mutant. Here, we presented that
different sets of genes are involved in N-rich and N-limited
oxidative stress conditions and NtrC-sensed nitrogen
availability is the most important prerequisite for full cellular
defense against oxidative stress in P. putida.
F004
Metaproteomics in Microbial Ecology
Jong-Shik Kim*, Jung-Hee Woo, Jun-Tae Kim,
Nyun-Ho Park, and Choong-Gon Kim
Gyeongbuk Institute for Marine Bioindustry
New technologies are providing unprecedented knowledge into
microbial community structure and functions. Even though
nucleic acid based approaches do provide a lot of information,
metaproteomics could provide a high-resolution representation
of genotypic and phenotypic traits of distinct microbial
communities. Analyzing the metagenome from different
microbial ecosystems, metaproteomics has been applied to
seawater, human guts, activated sludge, acid mine drainage
biofilm and soil. Although these studies provide different
approaches, they elucidated that metaproteomics could provide
a link between microbial community structure, function,
physiology, interaction, ecology and evolution. These
approaches are reviewed here to help gain insights into the
function of microbial community in ecosystems.
[This work was supported by The NCRC (grant #:
20090091491 and KSEF grant (R01-2008-000-10697-0).)]
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F005
F007
A Tip-to-tip Interaction Between AcrA and TolC Plays
an Essential Role for the Formation of Functional
Bacterial Tripartite Efflux Pump AcrAB-TolC
1*
2
2
Hong-Man Kim , Yongbin Xu , Shunfu Piao ,
Se-Hoon Sim1, Minho Lee1, Nam-Chul Ha2, and
Kangseok Lee1
1
Department of Life Science (BK21 program), Research Center for
Biomolecules and Biosystems, Chung-Ang University, 2Department
of Manufacturing Pharmacy, College of Pharmacy and Research
Institute for Drug Development, Pusan National University
Tripartite efflux pumps are involved in antibiotic resistance and
toxic protein secretion. In this study, we investigated the
assembly mechanism of the major multidrug efflux pump
AcrAB-TolC. Site-directed mutational analyses revealed that the
conserved residues located at the tip region of the α-barrel of the
membrane fusion protein (MFP) AcrA play an essential role in
the binding of an outer membrane factor (OMF) TolC, as well as
in the action of tripartite efflux pumps. Genetic complementation
experiments further confirmed that the AcrA hairpin tip region is
functionally related to the TolC aperture tip region. In addition,
we provided in vivo functional data showing that AcrA shares a
TolC binding mode with the hexameric MacA. Our results
demonstrate that a tip-to-tip interaction between AcrA and TolC
is required for the formation of the functional tripartite multidrug
efflux pump. This finding may offer a molecular basis for
understanding the multidrug resistance of pathogenic bacteria.
Synthesis of CMP-Neu5Ac Using Metabolically
Engineered Escherichia coli
Hwa Young Choi*, Seong kee Cho, Jeong Mi Park, and
Nam Soo Han
Chungbuk National University, Department of Food Science and
Technology
Cytidine 5'-monophosphate N-acetylneuraminic acid (CMPNeuAc) is an essential precursor for the synthesis of
sialyloligosaccharides. In order to produce CMP-NeuAc, we
employed a recombinant E. coli system which was engineered
by gene transformation and gene knock-out techniques. The
neuB gene was for the conversion of N-acetylmannosamine
(ManNAc) and phosphoenolpyruvate (PEP) to Neu5Ac, and
the neuA gene was for the production of CMP-Neu5Ac from
cytidine triphosphate (CTP) and NeuAc. Additionally, neuC
encodes an epimerase that catalyzes the formation of ManNAc
from UDP-GlcNAc. We constructed expression vector using
pETDuet™-1 for the continuous production of CMP-Neu5Ac.
pETDuet™-1 was designed for the coexpression of two target
genes, pET-BA, pET-BC, which were made by cloning from
each of neuB, neuA, neuC. The NeuAc synthase, CMPneu5Ac synthase and UDP-GlcNAc 2 epimerase were
successfully expressed in E. coli BL21 (DE3) star. We
determined the molecular weight of neuB, neuA neuC on SDSPAGE and they were 38kDa, 48kDa and 44kDa, respectively.
[This work was supported by grants from the 21C Frontier Microbial
Genomics and Application Center Program of the Korean Ministry of
Science and Technology and Seoul R&BD program (10550).]
F006
F008
The Tip Region of the MacA Alpha-Hairpin Is
Required for the Binding to TolC of the Escherichia
coli MacAB-TolC Pump
1*
2
2
Yongbin Xu , Se-Hoon Sim , Saemee Song ,
Shunfu Piao1, Hong-Man Kim2, Xiao Ling Jin1,
Nam-Chul Ha1, and Kangseok Lee2
1
Department of Manufacturing Pharmacy, College of Pharmacy
and Research Institute for Drug Development, Pusan National
University, 2Department of Life Science (BK21 program),
Research Center for Biomolecules and Biosystems, Chung-Ang
University
The tripartite efflux pump MacAB-TolC found in gramnegative bacteria is involved in resistance to antibiotics. We
previously reported the funnel-like hexameric structure of the
adaptor protein MacA to be physiologically relevant. In this
study, we investigated the role of the tip region of its α-hairpin,
which forms a cogwheel structure in the funnel-like shape of
the MacA hexamer. Mutational and biochemical analyses
revealed that the conserved residues located at the tip region of
the α-hairpin of MacA play an essential role in the binding of
TolC. Our findings offer a molecular basis for understanding
the drug resistance of pathogenic bacteria.
[This work was supported by grants from the 21C Frontier
Microbial Genomics and Application Center Program of the
Korean Ministry of Science and Technology and Seoul R&BD
program (10543).]
188
Poster Sessions
Identification of Differentially Expressed Genes in
Flammulina velutipes with Anti-Tyrosinase Activity
Jae-Yong Cho* and Sang-Yoon Kim
Department of Pharmaceutical Engineering, College of Health
Science, Sangji University
It was previously shown that fruiting bodies of the mushroom,
Flammulina velutipes, exert anti-tyrosinase activity by producing
a triacylglycerol characterized as 1′,3′-dilinolenoyl-2′-linoleoylglycerol (LnLLn). In this study, we provide evidence that
the mycelia of F. velutipes grown on glucose, but not on
glycerol, exhibits anti-tyrosinase activity. To identify genes
involved in the rate-limiting step(s) in the biosynthesis of
LnLLn by F. velutipes, a RT-PCR method that involves
annealing control primers (ACPs) was employed. By using 120
ACPs, a total of 84 differentially expressed genes (DEGs) in F.
velutipes mycelia with anti-tyrosinase activity were cloned and
sequenced. Basic Local Alignment Search Tool (BLAST)
searches revealed that 72 of the genes have known sequence
homology. Of these, the genes involved in the modification of
fatty acids and their assembly into triacylglycerol (TAG) were
selected and further quantified by real-time RT-PCR.
[This research was supported by a grant (Code #
20070401034021) from Biogreen 21 Program, Rural
Development Administration, Republic of Korea, and in part
by the Sangji University Research Fund 2009.]
F009
F011
SigR-RsrA System Is Conserved in Actinomycetes
as Specific Thiol-Oxidative Stress Sensor
Characterization of A-Type Scaffold Proteins of FeS Assembly System in Schizosaccharomyces pombe
Yoo-Bok Cho1*, Yong-Gyun Jung1, Min-Sik Kim1,
Seok-Hyun Hong1, and Jung-Hye Roe1,2
1
School of Biological Sciences, Seoul National University,
2
Institute of Microbiology, Seoul National University
Su-Jin Jung*, Kyung-Chang Lee, and Jung-Hye Roe
Lab. of Molecular Microbiology, School of Biological Sciences
and Institute of Microbiology, Seoul National University
Zinc-containing anti-sigma factors (ZAS) are widespread
bacterial proteins that regulate their cognate sigma factors in
response to a variety of environment stresses or stimuli. They
have characteristic, conserved, HX3CX2C motif that is
responsible for zinc co-ordination. RsrA from Streptomyces
coelicolor (ScoRsrA) is one of the best characterized ZAS
proteins and is known as a disulfide stress sensor. However, it
is not well known of the signature pattern that makes it a
distinct disulfide sensor. We constructed simple validation
system to determine the thiol-oxidative stress sensitivity of the
anti-sigma factors from the fact that SigR autoregulates its
expression upon thiol-oxidative stress through RsrA.
Comparing conserved amino acid profiles of the confirmed
sensitive ZAS proteins with those of insensitive ones, we
identified a region that is important for thiol-oxidative stress
sensing. Importance of each residue within this region needs be
confirmed experimentally. Inspection of gene database to
predict the presence of putative redox-sensing ZAS factors has
been done.
F010
Nisin-Controlled Gene Expression System in Leuconostoc mesenteroides ATCC 8293
Seungkee Cho1*, Hyun-Ju Eom2, and Nam Soo Han1
1
Department of Food Science and Technology, Chungbuk
National University, 2Department of viticulture & Enology,
University of California, USA
Lactic acid bacteria (LAB) have been used successfully to
express a wide variety of recombinant proteins, ranging from
flavor-active proteins to antibiotic peptides and oral vaccines.
The nisin-controlled expression (NICE) system is the most
common of the systems for production of heterologous
proteins in LAB. The Nisin-Controlled gene Expression
system (the NICE system) is an efficient and promising gene
expression system based on the autoregulation mechanism of
nisin biosynthesis in the Lactococcus lactis. In the NICE
system, the membrane-located histidine kinase NisK senses the
inducing signal nisin and autophosphorylates, then transfers
phosphorous group to intracellular response regulator protein
NisR which activates nisA promoter to express the
downstream gene(s).The plasmid pJH24 is a popular vector for
nisin-inducible expression of heterologous genes in lactic acid
bacteria, and it was successfully expressed in Leuconostoc
mesenteroides ATCC 8293.
Iron-sulfur (Fe/S) cluster proteins are ubiquitous and play
critical roles in diverse cellular processes such as enzyme
reaction, respiration, DNA replication, and gene regulation.
The process of forming Fe-S clusters is best studied in
Escherichia coli and Saccharomyces cerevisiae, but not much
in Schizosaccharomyces pombe. Especially we investigated the
A-type scaffold proteins which is E.coli IscA homologues (Isa1
and Isa2). The isa1 (SPCC645.03c) and isa2 (SPBC3B9.17)
genes have been initially found in S. pombe to suppress growth
defects of Δgrx5 mutant that lacks mitochondrial monothiol
glutaredoxin. Each of these genes is essential for the growth of
S.pombe, in contrast to S.cerevisiae where even the double
mutant is viable. Since Grx5 is involved in Fe-S assembly in
mitochondria, we investigated interaction of Isa1 and Isa2 with
other Fe-S assembly proteins by in situ fluorescence complementation analysis. We found that Isa1 and Isa2 interact with
several components of Fe-S assembly system and several other
Fe-S containing proteins in mitochondria.
F012
Conservation of Genes with Redox-Responsive
Promoters as Direct Targets of Sigma Factor SigR
for Thiol-Reactive Stress Response in
Actinomycetes
Ji-Sun Yoo1*, Min-sik Kim1, Yoo-Bok Cho1, Joo-Hong Park1,
Gi-Baeg Nam1, Yong-Gyun Jung1, Yann Dufour2,
Tim Donohue2, and Jung-Hye Roe1
1
Laboratory of Molecular Microbiology, School of Biological
Sciences, Seoul National University, 2Department of Bacteriology,
University of Wisconsin, USA
In Streptomyces coelicolor a specific sigma-antisigma pair
SigR-RsrA modulates response toward thiol-oxidative stresses,
which are sensed through reactive cysteines in RsrA. In this
study we performed a genome-wide screening of direct target
genes of SigR by ChIP-chip analysis. Upon diamide treatment,
significantly elevated binding of SigR was observed in the
promoter regions of 79 genes that transcribes up to 122 genes
when considering operon structures. These direct target genes
of SigR encode known and predicted proteins for thiol
homeostasis, sulfur metabolism, modulation of ribosome
functions, proteolysis, transcriptional regulation, transporters,
oxidorectases, co-factor biosynthesis, stress response, DNA
damage repair, etc. The consensus SigR binding promoter
sequence was used to predict target genes in 47 genomes of
actinomycetes that contain SigR-RsrA orthologues, such as
corynebacteria, mycobacteria, streptomycetes, rhodococci, etc.
A large portion of the predicted SigR regulon members were
conserved throughout 47 bacterial species and some were
limited more to the genus level. Conserved functions toward
thiol-reactive stresses among actinomycetes system are found.
189
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F013
F015
The Construction of Gene Disruption and Shuttle
Vectors Based on Antibiotic Selection for Sulfolobus,
the Model Organism of Crenarchaeota
*
Sungmin Hwang , Kyoung-Hwa Choi, and Jaeho Cha
Department of Microbiology, College of Natural Sciences, Pusan
National University
Sulfolobus acidocaldarius, the first isolated hyperthermophile,
is one of the best studied species of thermoacidophilic
organism. It can be grown under aerobic and heterotrophic
conditions at 77°C and pH 3. To establish facile selection of
mutants and effective expression of heterologous genes,
Sulfolobus target gene disruption vector and EscherichiaSulfolobus shuttle vector were constructed. In the gene
disruption analysis in S. acidocaldarius, the gene disruption
vector was constructed by substitution of the gene of interest
with a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase gene,resistant to thermostable antibiotic simvastatin,
and it was designed to overexpress by the promoter of major
heat shock chaperonin tf55α. In order to prevent the possibility
of homologous recombination between host genomic DNA
and endogenous vector DNA, the HMG-CoA reductase and
tf55α genes were adopted from S. solfataricus P2. For
Escherichia-Sulfolobus shuttle vector, the ori from Sulfolobus
infected virus was used to replicate autonomously. In addition,
tf55α promoter and HMG-CoA reductase gene of S.
solfataricus P2 were used as a strong promoter and selection
marker, respectively.
F014
Glyoxal Detoxification via NADPH Dependent
Reductases of E. coli
Changhan Lee*, Insook Kim, and Chankyu Park
Department of Biological Science, Korea Advanced Institute of
Science and Technology
Glyoxal and methylglyoxal are reactive carbonyl compounds
and accumulated in vivo through various pathways, such as
glycation, lipid peroxidation and DNA oxidation. Previously,
we studied the involvement of aldo-ketoreductases (AKRs) in
methylglyoxal detoxification. We also reported that YqhD,
NADPH dependent aldehyde reductase, is crucial in glyoxal
detoxification. Here, we assessed the role of various reductases
including AKRs (YqhE, YafB and YghZ) that are likely to be
involved in GO metabolism. Enzyme activities of the
reductases, including YqhD, were assessed by measuring
oxidation rate of NADPH. In addition, glyoxal sensitivity of
the mutant strains was compared by measuring inhibitory
concentrations. Expression levels of the genes induced by GO
were measured by real-time RT-PCR. The results imply that
GO can efficiently be detoxified by several different types of
NADPH-dependent redutases, i.e. AKRs and YqhD.
[Supported by the 21C Frontier Microbial Genomics and
Application Center Program]
F016
Construction of High Sensitive Detection System for
Endocrine Disruptors with Yeast n-Alkane-Assimilating Yarrowia lipolytica
The Expression of Sterigmatocystin Genes in
Aspergillus nidulans Is Controlled by flbF, a Gene
Required for Asexual Development
Eun-Min Cho* and Chi-Yong Eom
Seoul Center, Korea Basic Science Institute
Yong Jin Kim*, Yeong Man Yu, Sang Eun An, Sun-Ho Kim,
and Pil Jae Maeng
Department of Microbiology & Molecular Biology, College of
Biological Sciences and Biotechnology, Chungnam National
University
To construct a highly sensitive detection system for endocrine
disruptors (EDs), we have compared the activity of promoters
with the n-alkane-inducible cytochrome P450 gene (ALK1),
isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7),
and the translation elongation factor-1gene (TEF1) for
heterologous gene in Yarrowia lipolytica. The promoters were
introduced into the upstream of lacZ or hERα reporter gene,
respectively, and the activity was evaluated by β-galactosidase
assay by lacZ or western blot analysis by hERα. The
expression analysis revealed that the ALK1 and ICL1 promoter
were induced by n-decane and by EtOH, respectively. The
constitutive promoter of RPS7 and TEF1 showed significantly
high level of expression in the presence of glucose and
glycerol, respectively. Particularly, the TEF1 promoter showed
the highest β-galactosidase activity and a significant signal by
western blotting with the anti-estrogen receptor compared with
the other promoters.
Secondary metabolites, or biochemical indicators of fungal
development, are of intense interest to humankind due to their
pharmaceutical and/or toxic properties. Here, we present that
flbF, which encodes encode a potential transcription factor
with a C2H2 zinc finger DNA binding motif, bipartite nuclear
localization signal, and glutamine rich region, a gene shown to
control the Aspergillus nidulans asexual development, also
controls secondary metabolism. We generated a flbF deletion
mutant and analyzed the phenotype. As a result, flbF deletion
mutant showing delayed in the asexual development, suggesting that the flbF gene is required for asexual development in
A.nidulans, also accumulation of brown pigment on media,
suggesting that the flbF gene is involved in secondary
metabolism. Specifically, flbF regulates the expression of
genes implicated in the asexual development and synthesis of
the mycotoxin sterigmatocystin.
[This work was supported by grant from Korea Research
Foundation]
190
Poster Sessions
F017
F019
Glyoxal-Induced DNA Damage Involves the Recombinational Repair System
Jihong Kim*, Changhan Lee, and Chankyu Park
Department of Biological Science, Korea Advanced Institute of
Science and Technology
Glyoxal (GO) is a toxic and mutagenic aldehyde compound,
causing DNA damage by modifying guanine and cytosine or
by disrupting sugar-phosphodiester backbone. GO-sensitive
mutants were isolated from Escherichia coli MG1655 strain by
disrupting gene randomly with TnphoA-132 transposon. The
mutations were mapped and found in recA as well as recC
gene, by inverse-PCR and DNA sequencing. RecA is central to
the SOS-response, an error-prone DNA repair system. RecC is
a subunit of exonuclease that is required for the homologous
recombination after double strand break. The isolation of such
mutants indicates that the SOS repair system is somehow
involved in repairing DNA damage induced by GO. We also
tested whether other DNA repair systems play a role in GOinduced DNA damage, such as the nucleotide excision repair
(uvrB), mismatch repair (mutS), base excision repair (mutM,
mutY), and SOS repair (umuC, dinB, recF) systems. Effects of
other repair systems were much lower than that of RecAC,
confirming the major role of RecA-mediated SOS repair in
GO-induced cell survival as well as GO-induced mutagenesis.
[Supported by the 21C Frontier Microbial Genomics and
Application Center Program.]
Cloning, Sequencing, and Phylogenetic Analyses of
a Carbon Monoxide Dehydrogenase Gene Cluster
in Terrabacter carboxydivorans
Jae Ho Lee*, Sae Woong Park, and Young Min Kim
Department of Biology, Yonsei University
Carboxydobacteria are a group of bacteria which are capable of
growing aerobically on carbon monoxide as a sole source of
carbon and energy by using carbon monoxide dehydrogenases
(CO-DH) as a key enzyme for the oxidation of carbon monoxide
(CO). Terrabacter carboxydivorans, one of the carboxydobacteria, is able to grow aerobically on CO of low concentration as
a sole source of carbon and energy. It has been known that CODH in T. carboxydivorans has no immunological interactions
with those of other carboxydobacteria. This imply that CO-DH
in T. carboxydivorans might be divided into a new group of
CO-DHs. To identify this hypothesis, the three structural genes
of CO-DH in T. carboxydivorans were cloned, sequenced, and
analyzed. The genes were clustered in the transcriptional order
cutB-cutC-cutA. The cloned cutB, cutC, and cutA genes had
open reading frames of 894, 552, and 2,433 nucleotides, and
encoded 297, 183, and 810 amino acids, respectively. The
mean identities in the deduced amino acid sequences of the
CutA, CutB, and CutC of T. carboxydivorans with those of
other carboxydobacteria were 31.75%, 34%, and 48%, respecttively. The phylogenetic trees of the CutA, CutB, and CutC of
T. carboxydivorans showed that CO-DH in T. carboxydivorans
is a new group of CO-DH that has not been reported before.
[This study was supported by a grant from NRF [20090071521].]
F020
F018
Cadmium Regulates Expression of SML1 Which Is
a Rnr Inhibitor on the Post-Transcriptional Level
in Saccharomyces cerevisiae
*
In-Joon Baek and Cheol-Won Yun
School of Life Sciences and Biotechnology, Korea University
To understand the toxic mechanism of cadmium in living
organisms, we used Saccharomyces cerevisiae model system
and screened yeast mutants which are related to cadmium.
Δsml1, Δrnr3, Δwtm1, and Δwtm2 strains were found that
resistant to cadmium. In plate assay, Δwtm1, Δwtm2 and Δrnr3
strains were resistant to cadmium and expression level of
RNR3 was down-regulated in Δwtm1 and Δwtm2 strains. In
northern blot, there was no change in SML1 expression in WT,
Δrnr3, Δrnr1, Δwtm1, Δwtm2, Δdun1,and including deletion
strains of ESCRT subunits whether cadmium treated or not. It
is well known that sml1p is phosphorylated and degradation by
dun1p in DNA stress condition. However there were
remarkably increased translational level of SML1 in response
to cadmium stress in above strains. In plate assay, many
deletion strains of ESCRT subunits were sensitive to cadmium
stress. To find degradation pathway of sml1p, we performed
western blot with MG-132, proteasome inhibitor, in Δpdr5
strain that limit the efflux of protease inhibitor. Sml1p was not
degradation by DNA damage agents. These results imply that
cadmium has a different toxic mechanism from other DNA
damage agents such as MMS and HU.
Comparative Transcriptome Analysis of the Heat
Shock Response in Hansenular polymorpha
Hye-Yun Moon1,2*, Min jeong Shon3, Ohsuk Kwon3,
Jeong-Yoon Kim2, and Hyun Ah Kang1
1
Department of Life Science, Chung-Ang University,
2
Department of Microbiology, Chungnam National University,
3
Korea Research Institute of Bioscience and Biotechnology
Hansenula polymorpha is a thermotolerant methylotrophic
yeast that can grow up to 48°C. We carried out the
transcriptome analysis of two H. polymorpha strains, DL1 and
NCYC495, and observed more extensive change of expression
profiles in the DL1 strain with less thermotolerance than in the
NYCY495 under heat stress condition. Bioinformatics analysis
revealed the presence of heat shock elements in the promoter
region of commonly up-regulated genes in the both H.
polymorpha strains upon heat stress. Interestingly, some sets of
genes were differentially regulated for each strain of H.
polymorpha, implying that the detailed heat tolerance
mechanism of two strains might be significantly different.
Moreover, comparative transcriptome analysis showed that the
expression of a set of genes related to stress-response and cellwall integrity was already higher in the NCYC495 than in the
DL1 strain even at normal growth temperature, reflecting the
thicker cell wall structure and the higher tolerance to heat
shock of the NCYC495 strain.
[Supported bygrants from 21C Frontier Microbial Genomics
and Application Center Program and from NRF.]
191
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F021
F023
Global Expression Profile Analysis of Ethanol and
Heat Stress Responses in Saccharomyces cerevisiae
Identification of Putative Proteins Interacting With
Mating Type Proteins in Gibberella zeae
Jinho Choo*, Hye Yun Moon, Seon Ah Cheon, and
Hyun Ah Kang
Department of Life Science, Chung-Ang University
Eun Ji Cho1*, Hee-Kyoung Kim1, Yin-Won Lee2, and
Sung-Hwan Yun1
1
Department of Medical Biotechnology, Soonchunhyang
University, 2Department of Agricultural Biotechnology, Seoul
National University
For the development of bioethanol production process based
on lignocellulosic biomass, the yeast strains with increased
resistance to heat and ethanol stress are highly desired. In the
present study, we analyzed the change of global gene
expression profiles of Saccharomyces cerevisiae under three
conditions, such as 8% (v/v) ethanol stress, 39°C heat stress,
and the combined stress of ethanol and heat (8% ethanol at
39°C). Extensive alteration of gene expression was observed at
single stress condition of heat or ethanol. The commonly upregulated genes were mostly related to energy and cell stress
defense, whereas the commonly down-regulated genes were
involved in DNA processing, transcription, and protein
synthesis. Unexpectedly, the extent of gene expression change
was shown to be much less under the double stress condition
compared to under the single stress condition. We combined
the expression profiles with metabolic pathways to investigate
the common and unique features of metabolic remodeling in
the ethanol and heat shock responses, which would provide
new information applicable to the construction of multi stresstolerant yeast strains.
[Supported by a grant from NRF.]
F022
Gibberlla zeae is a self-ferile ascomycete with ubiquitous
geographic distribution, causing serious diseases in cereal
crops. We have focused on the mating-type (MAT) genemediated regulatory network which governs the sexual
developmental pathway in G. zeae. To identify putative
proteins that interact with MAT proteins, we have employed a
yeast two-hybrid assay using 4 different MAT gene products
as baits. So far, screening the fungal cDNA library obtained
from sexual development stage with MAT1-1-3 or MAT1-2
bait revealed 17 positive cDNA clones, for examples, those
encoding GTP cyclohydrolase 2 and MAT1-1-1. The former is
known to be involved in folic acid metabolism, and the latter is
a gene product encoded by MAT1-1 locus. In particular, the
identification of MAT1-1-1 from the prey library indicates that
both MAT1-1-3 and MAT1-1-1 proteins may undergo
heterodimerization for the transcriptional regulation of sexual
development-related genes in G. zeae. A more detailed
description of the genes obtained in this study will be presented.
[This work was supported by a grant (2009-0075256) from
NRF.]
F024
Effect of PyrR on the Expression of pyrH in
Corynebacterium glutamicum
Acyl Carrier Protein IacP Contribute to Salmonella
typhimurium Invasion of Epithelial Cell
Jae-Hyung Jo* and Hyune Hwan Lee
Department of Bioscience and Biotechnology, Hankuk University
of Foreign Studies
Jeong Seon Eom*, Jin Seok Kim, Jung Im Jang, and
Yong Keun Park
School of Life Science and Biotechnology, Korea University
The UMP kinase of Corynebacterium glutamicum, encoded by
pyrH, is the key enzyme in the biosynthesis of pyrimidine
nucleotides. It catalyzes the conversion of UMP to UDP. It was
reported that the expression of pyrH in Bacillus subtilus is
controlled by several factors such as UMP, UDP, UTP and
specially by the attenuation of the transcript of pyrH by
binding of PyrR. However, very little is known in the
mechanism of regulation of pyrH in Corynebacterium
glutamicum. Previously, we reported that the direct binding of
PyrR to the pyrH promoter regulates the expression instead of
attenuation in C. glutamicum. To further reveal the regulation
of pyrH by PyrR, pyrR was knocked-out and the effect on the
expression of pyrH was studied by real time PCR. As result,
the level of pyrH expression in pyrR knocked-out mutant is
two times higher than the wild type. This result is the evidence
of the regulation of pyrH expression PyrR in C. glutamicum.
Salmonella Pathogenicity Island 1(SPI-1) encodes a type III
secretion system(T3SS) that translocates effector proteins into
intestinal cells to promote invasion through actin cytoskeletal
rearrangements. The iacP gene belongs to the SPI-1 and is able
to be categorized into ACP family by sequence similarity,
however its function is poorly understood. We investigated the
role of IacP in Salmonella-induced invasion. Immunofluorescence microscopy of actin revealed that iacP mutant partially
impaired the actin rearrangement. The inositol polyphosphatase SopB is involved in invasion, Akt activation,
biogenesis of Salmonella-containing vacuole (SCV). Secretion
of SopB in iacP mutant was reduced and translocation and
membrane localization of SopB into INT407 cells were also
decreased. Furthermore, decreased secretion of SopB resulted
in inability of Akt activation and SCV maturation. And, we
identified the flagellar subunit which was upregulated in iacP
mutant from microarray data analysis and over secretion of the
flagellar subunit were also confirmed by LC/MS. These results
suggest that S.typhimurium IacP could modification of the
target protein to reinforce the invasion into host cell.
192
Poster Sessions
F025
F027
N-Terminal Signal of InvE is Required for Type III
Secretion of Salmonella Translocon SipB and SipD
Fission Yeast Homolog of Tho1p Is Involved in
mRNA Export
Jin Seok Kim*, Jung Im Jang, Jeong Seon Eom,
Dae Woo Kang, and Yong Keun Park
School of Life Sciences and Biotechnology, Korea University
Ye-Seul Cho*, Cha-Yeon Kim, Ae-Rhee Chae, and
Jin Ho Yoon
School of Biological Sciences and Chemistry, Basic Science
Research Institute, Sungshin Women’s University
Type III secretion system (T3SS) is composed of more than 25
proteins, which constitute three major part to inject the
virulence effector proteins into host cytoplasm ; basal body
embedded in bacterial membrane, polymerized needle, and
translocon connecting T3SS needle to host membrane.
Secretion of Salmonella translocon proteins, SipB, SipC and
SipD were regulated by InvE which is broadly localized in
cytoplasm and membrane, but not secreted into culture
supernatant. InvE has two coiled-coil domains in N-terminus
and C-terminus respectively and C-terminus coiled-coil
domain is important for SipB secretion.
We show here that secretion of SipB and SipD were blocked
when N-terminus 30 amino acids were deleted in InvE,
indicating that InvE N-terminus take SipB and SipD into T3SS.
The fact that N-terminal signal is characteristics of many type
III secreted proteins suggested that InvE has ancestral secretion
signal in N-terminus recognized by T3SS membrane protein.
[Supported by grants from KHIDI-A090891]
F026
In eukaryotes, nuclear export of mRNA takes place through the
nuclear pore complex (NPC) embedded in the nuclear
envelope and several soluble transport factors are involved in
this process. We constructed deletion mutants of fission yeast
Schizosaccharomyces pombe gene that encodes a protein
homologous to Tho1p in budding yeast Saccharomyces
cerevisiae, which is a conserved RNA binding nuclear protein
that specifically binds to transcribed chromatin in a THO- and
RNA-dependent manner and genetically interacts with the
shuttling hnRNP Nab2. The fission yeast tho1 gene encodes an
245 amino-acid protein with predicted molecular weight of
26.7 kDa. The tho1 gene is not essential for vegetative growth.
The accumulation of poly(A)+ RNA in the nucleus is exhibited
when expression of tho1 is repressed or overexpressed. And
functional Tho1-GFP signal was detected in nucleus. These
results suggest that tho1 in fission yeast is involved in nuclear
export of RNA.
[Supported by grants from Sungshin Women’s University]
F028
The Organization of Gene Cluster in the Length of
55 kb for the Biosynthesis of a Polyether Antibiotic,
Laidlomycin, from Streptomyces sp. KCTC 10631BP
Isolation of Synthetic Lethal Mutants with rsm1
Null Allele That Is Involved in mRNA Export in
Fission Yeast
Jae Yoon Hwang*, Nguyen Phan Kieu Hanh, and
Doo Hyun Nam
Faculty of Pharmacy, Yeungnam University
Yun-Seon Park*, DongGeRaMi Moon, and Jin Ho Yoon
School of Biological Sciences and Chemistry, Basic Science
Research Institute, Sungshin Women’s University
Laidlomycin, a polyether antibiotics produced by Streptomyces
sp. KCTC 10631BP, shows strong antimicrobial activity
against methicilin-resistant Staphylococus aureus (MRSA) and
vancomycin-resistant enterococci (VRE). A 708 bp PCR
fragment of epoxidase gene was used as a probe to screen the
cosmid library of S. sp. KCTC 10631BP constructed in
SuperCos-1 vector. Among cosmid clones, pSLD-36 gave a
strong signal by colony hybridization and was found to contain
some essential genes like type I polyketide synthase (PKS),
epoxidase and epoxide hydrolase genes involved in the
biosynthesis of polyether antibiotic, laidlomycin. By chromosome walking using enoyl reductase and epoxide hydolase I
probes, another cosmid clone named pSLD-BI5 encoding the
downstream region of PKS gene was further screened. The full
length of the sequenced laidlomycin biosynthetic gene cluster
was around 55kb. The putative gene cluster was highly
homologous with the biosynthetic gene cluster for a typical
type I PKS polyether antibiotic, monensin of S. cinnamonensis.
The organization of the cloned gene cluster will be discussed
in this presentation.
In order to identity the genes in fission yeast Schizosaccharomyces pombe that are functionally involved in mRNA export,
mutants that showed growth retardation or synthetic lethality
with the rsm1 null allele were isolated. The rsm1 null mutant is
not essential for growth but it showed a mild accumulation of
poly(A)+ RNA in the nucleus. For this screening, we used the
rsm1 null mutant harboring the pREP81X-rsm1 vector, where
the expression of rsm1 is repressed in the presence of thiamine.
This strain was mutagenized with EMS and approximately
320,000 colonies were analyzed. The mutant colonies that
showed growth defects only in the presence of thiamine were
screened at 27°C. Ten mutants were finally isolated in this
screening and tentatively named as SLrsm1 through SLrsm10.
Vectors containing the genes known to be involved in mRNA
export were transformed into theses SLrsm mutant cells and
checked whether these genes could complement the growth
defects of these mutants in the presence of thiamine. These
results suggest that SLrsms isolated in fission yeast interacted
genetically with rsm1 and were defective in mRNA export.
[Supported by grants from KRF]
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F029
F031
The Function of Novel Non-Coding RNA Against
Innate Immune System in Salmonella typhimurium
Isolation and Characterization of a HypoxiaSensitive Mutant UV7 in Aspergillus nidulans
Sin Yeon Kim*, Yong Heon Lee, and Yong Keun Park
School of Life Sciences and Biotechnology, Korea University
Chinbayar Bat-Ochir* and Suhn-Kee Chae
Department of Biochemistry and Center for Fungal Pathogenesis,
Paichai University
Salmonella are bacterial pathogens that have been well studied
on virulence mechanisms, pathogenesis, and many fundamental
pathways. While these investigations have focused on protein
functions, Salmonella have also the remarked mechanism for
RNA-mediated regulation. Bacterial non-coding RNAs were
transcribed in response to various stresses. We have searched
for a non-coding RNA as being part of stress responses
including pH, nitrosative stress and oxidative stress. Reactive
oxyzen species (ROS), reactive nitrogen species (RNS), and an
acidic stress function as strong antimicrobial substances in
macrophage innate immunity. In this study, we chose a mutant
that shows susceptibility to oxidative stress by transposon
mutagenesis. We show that the non-coding RNA is increased
at oxidative stress and stable throughout the time course like
other regulatory small RNA. Also, we are investigating target
gene related to mechanism of the novel transcript from
microarray. Our results revealed that this novel intergenic
transcript may promote intracellular survival within the
macrophage and mouse in vivo.
Pathogenic microorganisms must overcome numerous
obstacles to successfully colonize in a host after their infection.
One of these barriers is hypoxia. Several hypoxia-sensitive
mutants in Aspergillus nidulans were obtained by UV
mutagenesis in our lab. One of those mutants UV7 showed a
strong hypoxia-sensitive phenotype and high sensitivity to
itraconazole. A genomic DNA fragment complementing the
hypoxia-sensitivity of UV7 was isolated. One of ORFs within
the cloned genomic DNA complemented UV7. This gene was
named hosB (hypoxia-sensitive) and has 1,035 bp of ORF
encoding a polypeptide of 244 amino acids. A mutation UV7
was identified by DNA sequencing of hosB PCR products.
This indicated that the hosB gene was not an intergenic
suppressor. One putative conserved domain of unknown
function was found in HosB. A point mutation resulted in the
68th tyrosine into aspartic acid was occurred in this domain.
HosB had transmembrane domains and was suggested to be
localized in ER membrane. Null mutant of hosB showed the
hypoxia-sensitive phenotype as UV7 which was complemented by overexpressing of hosB under the inducible
promoter niiA.
[Supported by Grants from NRF]
F030
F032
Functional Analysis of the hitB Gene Required for
Hypoxic Growth in Aspergillus nidulans
Transcriptomic Response of the Methylotrophic
Yeast Hansenula polymorpha to Hydrogen Peroxide
Jun-Yong Kwak* and Suhn-Kee Chae
Department of Biochemistry and Center for Fungal Pathogenesis, Paichai University
Eun Hye Kim1,2*, Min Jee Kim1, Doo-Byoung Oh1,
Jeong-Yoon Kim2, Hyun Ah Kang3, and Ohsuk Kwon1
1
Integrative Omics Research Center, Korea Research Institute of
Bioscience and Biotechnology 2Department of Microbiology,
Chungnam National University, 3Department of Life Science,
Chung-Ang University
Sterol-regulatory element binding protein (SREBP) is an ER
membrane tethered transcription factor that controls synthesis
of cholesterol and fatty acid in mammalian cells. Previously,
we showed that HitA an A. nidulans SREBP homolog was
shown to be essential for hypoxic growth. In this study, the
hitB gene encoding a polypeptide showing similarities to the
N-termini but lacking the transmembrane regions and the Ctermini found in HitA and SREBPs was analyzed. hitB null
mutants failed to grow in hypoxic condition in similarly shown
to hitA null mutants. The N-terminus of HitA or HitA
complemented the hypoxia-sensitive phenotype of ∆hitB,
while the hypoxia-sensitive phenotype of ∆hitA was not
complemented by HitB in hypoxia. We further confirmed
direct binding of HitA to the promoter of hitB by the ChIP
assay, while HitB failed to bind to the hitA promoter. HitB
interacted with the HitA N-terminus to form heterodimer as
well as with HitB in in vitro resin binding assay. HitB was
localized to the nucleus based on the observation of HitB-RFP
fusion proteins with a fluorescence microscope.
[Supported by Grants from NRF]
In the methylotrophic yeast Hansenula polymorpha, methanol
is first oxidized to formaldehyde by a peroxisomal alcohol
oxidase, generating high levels of hydrogen peroxide. H.
polymorpha can tolerate oxidative stress, but the global
oxidative stress response has not been elucidated. In this study,
the genome-wide expression profiles of H. polymorpha in
response to exposure to H2O2 were analyzed. About 50 genes
were either up-regulated or down-regulated more than twofolds in the presence of H2O2. The majority of the up-regulated
genes were involved in the oxidative stress response and
general stress response. On the other hand, the majority of the
down-regulated genes were involved in glucose utilization, cell
wall metabolism, and ribosomal protein synthesis. When
compared to the previously reported H2O2-induced transcriptmic responses of S. cerevisiae and S. pombe, similar subsets of
genes were also differently expressed in H. polymorpha.
However, the number of differently expressed genes was much
smaller clearly reflecting its relatively high tolerance to
oxidative stress.
[Supported by KRIBB Research Initiative Program grant and
Korea Research Foundation grant (No. 2009-0075186).]
194
Poster Sessions
F033
F035
Transcriptome Analysis of Xylose Metabolism in
the Theromotolerant Methylotrophic Yeast Hansenula polymorpha
Characterization of the CpxAR Two-Component
Signal Transduction System of the Capnophilic
Rumen Bacterium Mannheimia succiniciproducens
Oh Cheol Kim1,2*, Surisa Suwannarangsee1, Doo-Byoung Oh1,
Jeong-Yoon Kim2, Hyun Ah Kang3, and Ohsuk Kwon1
1
Integrative Omics Research Center, Korea Research Institute of
Bioscience and Biotechnology, 2Department of Microbiology,
Chungnam National University, 3Department of Life Science,
Chung-Ang University
Seulgi Yun1,2*, Eun-Gyeong Lee1, Won Seok Jung1,
Doo-Byoung Oh1, Sang Yup Lee3, and Ohsuk Kwon1,2
The thermotolerant methylotrophic yeast Hansenula polymorpha
is naturally capable of alcoholic fermentation of xylose, a
pentose sugar abundant in lignocellulosic biomass, even at high
temperature up to 48°C. In the present study, the transcriptomes
of H. polymorpha grown on xylose were compared with those of
glucose-grown cells both under aerobic and microaerobic
conditions. About two percent of the 5,848 H. polymorpha genes
were either up-regulated or down-regulated more than two-fold
during growth on xylose. The majority of the up-regulated genes
were involved in metabolism. Some genes involved in xylose
metabolism such as TAL2, XYL1 and GRE3 were also upregulated, even though their induction levels were only about
three folds. On the other hand, the majority of the downregulation genes were involved in metabolism and cellular
transport. Interestingly, some genes involved in glycolysis and
ethanol fermentation were also repressed during growth on
xylose, suggesting that these genes might be good targets for
engineering H. polymorpha to improve xylose fermentation.
[Supported by KRIBB Research Initiative Program grant and
Korea Research Foundation grant (No. 2009-0075186).]
1
Integrative Omics Research Center, Korea Research Institute of
Bioscience and Biotechnology, 2University of Science & Technology,
3
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, Center for
Ultramicrochemical Process Systems, and Department of BioSystems,
BioProcess Engineering Research Center and Bioinformatics Research
Center, Korea Advanced Institute of Science and Technology
The cpxR and cpxA genes of Mannheimia succiniciproducens
encode, respectively, proteins having about 70% and 50% of amino
acids sequence homology with CpxR response regulator and CpxA
sensor kinase of the bacterial Cpx two-component system involved
in envelop stress response. The purified N-terminally truncated
CpxA which was deleted for its transmembrane domain was able to
autophosphorylate and transphosphorylate CpxR, demonstrating that
these two proteins are a functional sensor kinase and a response
regulator, respectively. We constructed a cpxR mutant strain and
examined its phenotype under various growth conditions. The cpxR
mutant exhibited growth retardation in the presence of 0.2 mM H2O2
or 0.2 M NaCl. In addition, when putative targets were searched by
employing transcriptome analysis to overexpression of CpxR, many
genes involved in cell wall biosynthesis were differentially expressed.
Our data clearly indicate that CpxAR system of M. succiniciproducens might be involved in the envelop stress signaling.
[Supported by the Genome-based Integrated Bioprocess Project
grant of the Ministry of Education, Science and Technology
(MOEST) through the Korea Research Foundation (KRF).]
F034
Transcriptomic Response of Escherichia coli Host
to Fosmid Metagenome Library
Mun-Kyoung Min1*, Min Jee Kim1, Jae Jun Song2,
Doo-Byoung Oh1, and Ohsuk Kwon1
1
Integrative Omics Research Center, Korea Research Institute of
Bioscience and Biotechnology, 2Microbial Fusion Technology
Research Center, Korea Research Institute of Bioscience and
Biotechnology
To explore the effect of metagenomic DNA on the genomewide expression in Escherichia coli, we compared the
transcriptome profiles of E. coli transformed with the
metagenomic DNA libraries to those of untransformed E. coli.
When a fosmid library was transformed into E. coli EPI300
strain, 40 and 60 genes were up-regulated and down-regulated
by at least twofold, respectively. The majority of up-regulated
genes upon fosmid transformation were functionally grouped
into those responsible for transport and binding proteins. In
contrast, the majority of down-regulated genes were
functionally categorized into energy metabolism. Further,
when we compared changes in transcriptome profiles of E. coli
upon transformation with different fosmid metagenomic
libraries, similar genes were shown to be differentially
expressed in both transformants. Thus, our transcriptome data
will provide useful information to understand the genome-wide
response of E. coli to metagenome library and to develop an
efficient host system for metagenome cloning and functional
expression for activity-based screening.
[Supported by Bio R&D Programs of the Korea Research
Foundation (KRF).]
F036
Characterization of the NarPQ Two-Component
Signal Transduction System of the Capnophilic
Rumen Bacterium Mannheimia succiniciproducens
Eun-Gyeong Lee1,2*, Ju-young Kim1, Doo-Byoung Oh1,
Seon-Won Kim2, Sang Yup Lee3, and Ohsuk Kwon1
1
Integrative Omics Research Center, Korea Research Institute of
Bioscience and Biotechnology, 2Division of Applied Life Science (BK21),
EBNCRC and PMBBRC, Gyeonsang National University, 3Metabolic
and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering, Center for Ultramicrochemical Process Systems, and Department of BioSystems, BioProcess
Engineering Research Center and Bioinformatics Research Center,
Korea Advanced Institute of Science and Technology
The putative narP and narQ genes of Mannheimia succiniciproducens encoding, respectively, proteins having about 90% and 70%
of amino acids sequence homology with NarP response regulator
and NarQ sensor kinase were identified. To determine its target
operons, we analyzed the genome-wide transcriptome profiles of M.
succiniciproducens in response to overexpression of NarP response
regulator. Our data showed that about 20 genes and 150 genes were
respectively up-regulated and down-regulated more than two-folds.
Interestingly, the expression of the putative napF gene encoding
periplasmic nitrate reductase was most strongly induced. A
transcriptional fusion Φ(napF’-lacZ) was constructed between the
promoter of napF and lacZ gene and introduced into an M.
succiniciproducens lacZ mutant strain to analyze its expression
under various culture conditions. The expression of Φ(napF’-lacZ)
was strongly induced in the presence of nitrate, suggesting that the
expression of napF gene is regulated by a nitrate dependent manner.
[Supported by the Genome-based Integrated Bioprocess Project
grant of the Ministry of Education, Science and Technology
(MOEST) through the Korea Research Foundation (KRF).]
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F037
F038
Characterization of Plasmid pSY1 in Sphingobium
chungbukense DJ77
Nucleotide Sequence and Secondary Structure of
23S rRNA from Sphingobium chungbukense DJ77
Mun-Sik Shin1, Kyung-Bae Min2, Ji-Eun Yu2, Hye-Jin Bae2
and Young-Chang Kim1,2,3*
1
Department of Synthetic Biology, Chungbuk National University,
2
Department of Microbiology, Chungbuk National University,
3
Biotechnology Research Institute, Chungbuk National
University
Mun-Sik Shin1, Won-Ho Lee2, Joo-Han Gwak2,
Woo-Yeoung Jang2, and Young-Chang Kim1,2,3*
1
Department of Synthetic Biology, Chungbuk National University,
2
Department of Microbiology, Chungbuk National University,
3
Biotechnology Research Institute, Chungbuk National University
Sphingobium chungbukense DJ77, isolated in our laboratory, is
an aerobic gram-negative bacterial strain. This strain is able not
only to degrade aromatic hydrocarbons, such as phenanthrene,
anthracene, biphenyl, salicylate, p-hydroxybenzoate and
benzoate, but also to produce exopolysaccharides, which
possess an increasing importance in the food industry. This
study determined the complete nucleotide sequence of plasmid
pSY1 from S. chungbukense DJ77. It was 402,272 bp long
with a G+C content of 61.81%. The 362 open reading frames
(ORFs) were found. We predicted these ORFs would encode
proteins associated with plasmid replication, transcription,
conjugation of genes, plasmid stability/partition, hypothetical
protein, and some other functions. Besides these genes, the
plasmid contained Phn genes for aromatic hydrocarbon
degradation. No other plasmid homologous to pSY1 in overall
nucleotide sequence or gene organization could be found in the
NCBI database.
196
Poster Sessions
Sphingobium chungbukense DJ77 is a Gram-negative bacterium
that is a very interesting organism due to its capacities to
degrade monocyclic and polycyclic aromatic compounds,
synthesizing glycosphingolipids as components of the cell
envelope, and producing exopolysaccharides as extracellular
polymers. Three 23S ribosomal RNA genes from the Sphingobium chungbukense DJ77 have been sequenced. All the three
sequences are the same. The secondary structure of the 2796base-long RNA was proposed. We made the secondary
structure of the 23S rRNA based on E. coli model and found
eight specific regions. We found the variable regions in
Sphingomonads, NovoSphingobium aromaticivorns, Sphingomonas wittichii, Sphingopyxis alaskensis, and Sphingobium
chungbukense DJ77.
G001
G003
Solvent/Detergent Process for the Manufacture of
Porcine Acellular Dermal Matrix (ADM): Efficacy
of Decellularization and Virus Inactivation
Identification of a Multicopy Suppressor Gene
Complementing the Arginine-Auxotrophic argJ
Mutation in Corynebacterium glutamicum
Jung Eun Bae1*, Dong Hyuck Lee1, Jeong Im Lee1,
Eun Kyo Jeong1, Jae Il Lee1, Da Mi Choi2, Jin Young Kim2,
Jae Hyoung Ahn2, and In Seop Kim1
1
Department of Biological Sciences, Hannam University,
2
Hans Daedeok R&D Center, Hans Biomed Corp.
Jae-Yong Cho* and Gui-Hye Hwang
Department of Pharmaceutical Engineering, College of Health
Science, Sangji University
Human ADM has been used in the management of fullthickness injuries as well as full-thickness burns. Human ADM
is currently produced by decellularization from human cadaver
skin in special buffered solutions. It retains intact basement
membrane complex, collagen bundle architectures and the
dermal vasculatures. However human viral contamination of
donated skin as well as finding suitable donors are major
obstacles to produce human ADM in a timely manner. To
address these shortcomings, the manufacturing process for
acellular xenograft dermal matrix using porcine skin has been
developed. The most critical process for porcine ADM is to
remove porcine immunogenic cells while maintaining the
integrity of the nonimmunogenic components of xenograft
dermis. Decellularization process using a solvent/detergent
combination (0.1% TnBP/2% deoxycholic acid) has been
developed. This process is also effective in inactivating porcine
enveloped viruses such as pseudorabies virus, porcine epidemic
diarrhea virus, and porcine rotavirus.
[Supported by Business for Cooperative R&D between
Industry, Academy, and Research Institute funded Korea Small
and Medium Business Administration in 2008-2009]
G002
We recently proposed a metabolic engineering strategy for Lornithine production based on the hypothesis that an increased
intracellular supply of N-acetylglutamate may further enhance
L-ornithine production in a well-defined recombinant strain of
Corynebacterium glutamicum. In the present work, an argJ–
deficient arginine auxotrophic mutant of C. glutamicum is
suppressed by a different locus of C. glutamicum ATCC13032.
Overexpression of the NCgl1469 open reading frame (ORF),
exhibiting N-acetylglutamate synthase (NAGS) activity, was
able to complement the C. glutamicum arginine-auxotrophic
argJ strain and showed the increased NAGS activity from 0.03
to 0.17 units mg-1 protein. Additionally, overexpression of the
NCgl1469 ORF resulted in a 39% increase in excreted Lornithine. These results indicate that the intracellular supply of
N-acetylglutamate is a rate-limiting step during L-ornithine
production in C. glutamicum.
[This research was supported by the Advanced R&D
Supporting Business between Industry and University funded
by the Small and Medium Business Administration, Republic
of Korea, and in part by the Sangji University Research Fund
2009.]
G004
Hydrogen Peroxide Treatment as an Effective
Measure for Enhancing Viral Safety of Allo Bone
Transplants
Genetically Engineered Salmonella typhimurium as
a Drug Delivery System for Interferon-Gamma
Induced Therapy Against Melanoma
Jeong Im Lee1*, Jung Eun Bae1, Eun Kyo Jeong1,
Jae Il Lee1, Dong-Joo Yu1, Seon Hyun Jeon2, Ji-Hwa Chae2,
Jung Sun Jeong1, and In Seop Kim1
1
Department of Biological Sciences, Hannam University, 2Hans
Daedeok R&D Center, Hans Biomed Corp.
Won Suck Yoon1,2*, EunJae Kim3, Ji-hyeon Choi3, and
YongKeun Park3
1
School of Life Sciences and Biotechnology, Korea University,
Seoul, Republic of Korea, 2Office of Research, Korea University,
Seoul, Republic of Korea, 3School of Life Sciences and
Biotechnology, Korea University
Human demineralized bone matrix (DBM) has been clinically
used as it is osteoinductive. The ability to remove and/or
inactivate known and potential viral contaminants during the
manufacturing process of DBM has become an important
parameter in assessing the safety of the products. In order to
increase the safety of DBM, 3% hydrogen peroxide treatment
process has been applied. Viral clearance validation method to
evaluate efficacy of 3% hydrogen peroxide was developed and
then the effect of the treatment in inactivating viruses was
kinetically determined. A variety of experimental model
viruses for human pathogenic viruses, including the human
immunodeficiency virus (HIV), hepatitis A virus (HAV),
bovine herpes virus (BHV), bovine viral diarrhea virus
(BVDV), and porcine parvovirus (PPV) were selected for this
study. All the enveloped viruses such as HIV, BHV, and
BVDV were completely inactivated to undetectable levels in 1
hour treatment. Non-enveloped viruses such as HAV and PPV
were also very sensitive with the log reduction values of 1.58
and 4.21, respectively.
Salmonella has been used experimentally as an anticancer
agent because it shows selective growth in tumors. In this study,
we exploited genetically-engineered Salmonella typhimurium
expressing interferon-gamma (IFN-γ) as a tumoricidal agent to
enhance its therapeutic efficacy. IFN-γ was fused to SipB and
recombinant IFN-γ was produced and secreted into culture
supernatants. Attenuated S. typhimurium expressing recombinant
IFN-γ, invaded and induced the lysis of tumor cells, and
activated NK cells. Furthermore, when it was subcutaneously
administered into mice bearing melanomas, S. typhimurim
expressing IFN-γ, significantly inhibited tumor growth by 80%
(eight of ten mice were cured of cancer) and prolonged the
survival of the mice without inducing permanent immunity
against tumors. These results suggest that tumor-targeted
therapy using Salmonella expressing IFN-γ has potential for
the treatment of cancer.
[Supported by MKE and KOTEF through the Human
Resource Training Project for Strategic Technology]
197
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G005
G007
A Novel Method for the Detection of Viral Protein
NS5B Using a Streptavidin-Tagged RNA Oligonucleotide
Exploration of Biocatalytic Potential of Epoxide
Hydrolases from Marine Bacteria by Whole Genome
Analysis
Changhyun Roh* and Sung-Kee Jo
Korea Atomic Energy Research Institute
Jung-Hee Woo1*, Young-Ok Hwang2, Ji-Hyun Kang2,
Jangcheon Cho3, Jarone Pinhassi4, Sung Gyun Kang2, and
Sang-Jin Kim2
1
Gyeongbuk Institute for Marine Bio-Industry, 2From the Marine
Biotechnology Research Centre, Korea Ocean Research &
Development Institute, 3Department of oceanography, Inha
University, 4Marine Microbiology, Department of Pure and
Applied Natural Sciences, University of Kalmar, Sweden
The monitoring of viral protein NS5B has been considerable
interest of in developing simple and reliable methods for
detection the hepatitis C virus (HCV) for applications in
diagnostic medicine. At present, a variety of assay methods
have been developed for the qualitative and quantitative
detection of HCV. Generally, antibody is the most common
reagent in enzyme-linked immunosorbent assay and western
blotting for the detection of such specific proteins in samples.
The detection limit using antibody is approximately μmol level.
To use antibody bring problem such as being temperaturesensitive, specific reactions condition and requiring secondary
antibody conjugated with enzyme and fluorescent dye. To
overcome these bottlenecks, aptamers are introduced as a
substitute for antibody in the application of biosensors for
detection and measurement of biological or environmental
molecules. In this study, we designed a streptavidin-biotin
conjugation method, that is, the RNA oligonucleotide sensor
system that could quantify viral protein in solution with
streptavidin-tagged aptamer. The detection level was fmol
level with low affinity interactions in a novel designed system.
Marine organisms, in particular, represent great phylogenetic
diversity, making them reservoirs of unique genetic information and important natural resources for possible development.
As analysis of various genomic databases, one or more putative
epoxide hydrolase genes of sequenced organism were identified
about 10% of marine source. Multiple sequence alignments of
epoxide hydrolase and putative epoxide hydrolase genes can be
classified into three groups with subgroup. The known, putative
epoxide hydrolases from marine sources were tested for activity
towards various epoxide substrates using a GC analysis assay.
Using this assay, it appeared that of the 11 EHases that were
tested, 11 were active with one or more epoxides. These results
demonstrate that a couple of epoxide hydrolases have the
possible application as an industrial biocatalyst for the
production of enantipure epoxides or vicinal diols.
[This work was supported by the Marine and Extreme Genome
Research Center Program, Ministry of Land, Transport and
Maritime Affairs, Republic of Korea.]
G006
G008
Peptide Directed Synthesis of Silica Coated Gold
Nanocables
Screening of an Enantioselective Epoxide Hydrolase
from PAH-Degrading Bacteria
Jungok Kim1*, Nosang V. Myung2, and Hor-Gil Hur1
1
Department of Environmental Science and Engineering and
International Environmental Research Center, Gwangju Institute
of Science and Technology, 2Department of Chemical and
Environmental Engineering and Center for Nanoscale Science
and Engineering, University of California at Riverside
Jung-Hee Woo*, Tae-Hyung Kwon, Jong-Shik Kim,
Nyun-Ho Park, Jun-Tae Kim, Sun-Mee Hong, and
Choong-Gon Kim
Gyeongbuk Institute for Marine Bio-Industry
Here we report a biomimetic peptide directed method to
synthesize one-dimensional co-shell nanocables by sequentially guide formation of silica insulating shell on metallic gold
nanoribbons in aqueous and ambient conditions. In our
previous study, we have shown that gold nanoribbon was
fabricated by gold-synthesizing peptide, Midas-11, isolated
from phage-displayed peptide library. Furthermore, the silicasynthesizing peptide Si#6-C, which was designed and modified
from peptide R5 found in natural diatom, was used for binding
onto gold nanoribbon surfaces and the production of silica
shell. TEM, SEM, AFM, and fluorescence microscopic
analysis clearly indicate the formation of evenly coated silica
on gold nanoribbons regardless of the shapes of template cores
and thiol group attached with peptide is necessary for the
binding and forming amorphous silica coating. This is
significant since it demonstrated the ability to fabricate
functional nano-electronic component using a biomimetic
method.
[This work was supported by grants from the Research Center
for Biomolecular Nanotechnology at GIST, Korea, and the
21C Frontier Microbial Genomics and Applications Center
Program.]
198
Poster Sessions
Enantioselective synthesis or hydrolysis is getting much more
attention due to current concerns about mixed chirality of a lot
of chemical, pesticides and medicine. To screen strains
producing an epoxide hydrolase (EHase) which hydrolyzes (R)
or (S)-epoxide preferentially, 7 strains of PAH-degrading
bacteria isolated from oil-contaminated sediment and commercial gasoline primarily by the capability of living on styrene
oxide were tested for EHase activity using gas chromatography
(GC). Among those, one strain was selected by highly
enantioselective hydrolysis of styrene oxide, confirmed by GC.
The EHase from one strain preferentially hydrolysed the (R)epoxide of styrene oxide, with a value of 98% ee (enantiomeric
excess). This study presents a first example which discovered
an enantioselective epoxide hydrolase from PAH-degrading
bacteria successfully.
[This work was supported by the Marine and Extreme Genome
Research Center Program, Ministry of Land, Transport and
Maritime Affairs, Republic of Korea.]
G009
A Cell-Free Protein Producing DNA-Polymer
Hydrogel
Young Hoon Roh*, Nokyoung Park, and Dan Luo
Cornell University
A Cell-Free Protein Producing DNA Hydrogel Proteins are
important biomaterials and are generally produced in living
cells. Here, we show a novel DNA hydrogel that is capable of
producing functional proteins without any living cells. This
protein-producing gel (termed ‘the P-gel system’ or ‘P-gel’)
consists of genes as part of the gel scaffolding. This is the first
time that a hydrogel has been used to produce proteins. The
efficiency was about 300 times higher than current, solutionbased systems. In terms of volumetric yield, the P-gel produced up to 5 mg ml-1 of functional proteins. The mechanisms
behind the high efficiency and yield include improved gene
stability, higher local concentration and a faster enzyme
turnover rate due to a closer proximity of genes. We have
tested a total of 16 different P-gels and have successfully
produced all 16 proteins including membrane and toxic
proteins, demonstrating that the P-gel system can serve as a
general protein production technology.
G011
Enhanced Butyric Acid Production and Resistance
to Sodium Butyrate by Clostridium tyrobutyricum
Mutant
Ki-Yeon Kim*, Youngsoon Um, and Byoung-In Sang
Clean Energy Center, Korea Institute of Science and Technology
To improve butyric acid production, Clostridium tyrobutyricum mutant LR7B4 was selected after repeated mutagenesis of
the parent strain Clostridium tyrobutyricum ATCC 25755 with
N-methyl-N’-nitro-N-nitrosoguanidine followed by an selective
enrichment on sodium butyrate. The selected mutant was
fivefold more resistant to sodium butyrate than the parent
strain. In batch culture on 120 g of glucose per liter, the mutant
produced more than 30 g/L butyric acid under pH control using
NaOH in bioreactor. Compared with the parent strain, this
mutated strain produced 14.3% more butyric acid and 18.5%
less acetic acid in 96 hours under the same culture conditions.
This result suggested that mutagenesis with N-methyl-N’-nitroN-nitrosoguanidine together with selective enrichment on
sodium butyrate improved production of butyric acid from
glucose in the fermentation.
[Supported by NYSTAR Faculty Development Program
Award, NYSTAR CAT grant, US National Science
Foundation's CAREER award (grant number: 0547330) and a
USDA NRI grant.]
G010
Properties of Bacillus licheniformis B1 β-1,4Glucanase Overproduced in Escherichia coli
Han Bok Kim*, Hye Jung Song, Hwang Yeon Kim,
Jae Sung Hwang, and Cho Hee Lee
Department of Biotechnology, The Research Institute for Basic
Sciences, Hoseo University
The Bacillsus licheniformis B1β-1,4-glucanase gene was overexpressed in Esherichia coli BL21. A soluble protein with a
mass of 50 kDa was overproduced. A protein having a mass of
37 kDa was secreted from B. licheniformis. It is likely that the
β-1,4-glucanase produced in E. coli contained the leader
peptide and unprocessed carboxy-terminal region, but its
processing occurred in the carboxy-terminal in Bacillus. The
optimal temperature of β-1,4-glucanase was 40°C and the
enzyme still had 76% maximal activity at 60°C. Although he
optimal pH of the enzyme was 7, the enzyme retained
considerable activities over the broad pH range. Acidic fungal
cellulases are frequently used in food, detergent, pulp, textile
industries. However, studies about neutral and alkaline
cellulase are limited. The β-1,4-glucanase developed in this
study might be useful for industrial applications in the fields of
biofuel development.
G012
Cloning and Expression of Indole Oxygenase Gene
Derived from Corynebacterium glutamicum in
Escherichia coli
Sisi Patricia Efkaen* and Jinho Lee
Department of Food Science & Biotechnology, Kyungsung
University
A putative gene derived from Corynebacterium glutamicum
was confirmed for its indole oxygenase activity. This gene was
cloned into the shuttle vector, pCES208 in Escherichia coli
under the regulation of tac promoter, and designated as pCOX1.
The complete open reading frame of this indole oxygenase was
1,413 bp long, which encodes a protein of 470 amino acids.
Crude extract of E. coli W3110/pCOX1 was prepared and
subjected to SDS-PAGE analysis. A band corresponding to
molecular mass of about 54 kDa was appeared and this result
correlated with the predicted molecular mass of the cloned
indole oxygenase. The E. coli harboring pCOX1 showed blue
color colony in LB plate. The pigment showing blue color was
prepared from E. coli/pCOX1, and identified as indigo by
experiments using UV spectrophotometer, TLC, and HPLC.
The enzyme activity of the indole oxygenase from the whole
cell of E. coli/pCOX1 cultured at LB medium with 0.1 M
IPTG showed 2.347nmol/min/mg DCW (dry cell weight).
Also, E.coli W3110 containing pCOX1, produced about
281mg/L of indigo after 48 hours cultivation in LB medium
with 5 g/L of tryptophan.
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G013
Diversity of Polyketide Synthase Genes in Lichen
Cladonia spp.
Hyun-Ju Noh1,2*, Jin sung Lee1, Chae Haeng Park1,
Eung-Soo Kim2, and Soon Gyu Hong1
1
Polar BioCenter, Korea Polar Research Institute, Korea Ocean
Research and Development Institute, 2Department of Biological
Engineering, Inha University
Lichens are well known to produce a great variety of
secondary metabolites including polyketides which have
diverse biological roles and potential uses in pharmaceutical
application. To attain a comprehensive understanding of
polyketide synthase (PKS) gene diversity of lichen species
from polar regions, forty-two Cladonia samples were selected
from Chile, the Artic and the Antarctic regions. The βketosynthase (KS) domains of putative PKS genes were
amplified and sequenced using degenerate primers. We
obtained 25 KS sequence fragments from direct sequencing
and 33 fragments from cloning. Phylogenetic analyses of 58
KS sequences have shown that 10 distinct PKS types were
retrieved, of which all belonged to non-reducing (NR) PKS.
Three and six types were included in NR clade I and clade II,
respectively. The last type of PKS was related to NR type I and
type II, but the specific relationship was not resolved well. The
most abundant PKS type consisted with 30 sequences from 23
samples in NR clade II. The sample with the most diverse PKS
types was included Cladonia furcata. It contained 8 types of
PKS, two of which belonged to NR clade I and the other six
types belonged to NR clade II.
G014
Screening of Garlic Fermentative Bacteria Using
Isolation Medium Containing Garlic Juices as Sole
Carbon and Nitrogen Sources
Su-Ok Kim1*, Jae-Won Park1, Mi-Young Won1,
Sang-Buem Cho1, Chan-Gul Lee2, and Soo-Ki Kim1
1
Department of Animal Sciences and Environment, Konkuk
University, 2Chodae F&P Co. Ltd.
Historically, garlic (Allium sativium) has been regarded as
protective food throughout the countries. The effectiveness of
garlic in cancer, aging and cardio-vascular disease have been
researched and free radical scavenging activity was regarded as
the most promising characteristics of garlic. Sulfuric compounds
in garlic are known as representative to antioxidant activity.
However, their low stability during processing such as washing
and packing are a problematic to manufacturing of garlic
product. To ensure the stability of their antioxidant activity,
fermentation has been investigated. In this study, the bacteria
that involved in garlic fermentation were screened. Grinded
fresh garlic juice was used as sole carbon and nitrogen sources
in isolation medium. Sodium acetate also used as an ingredient
to stabilize cytoplasmic pH. Finally grinded garlic juice,
sodium acetate, dipotassium phosphate, magnesium sulfate and
sodium chloride were included in isolation medium. From
isolation medium, one hundred eight strains were isolated and
thirty strains were selected using relative performance index
including both of antioxidant activity and viability.
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Poster Sessions
G015
Biochemical Characterization of β-Glucosidase
Produced by Phoma sp. Isolated from the Surface of
Rotten Tangerine Peel
Jung Youn Choi1,2*, Ah Reum Park1, Yong Jin Kim1,
Chang-Jun Cha2, and Jeong-Jun Yoon1
1
Green Process R&D Department, Green Chemistry &
Manufacturing System Division, Korea Institute of Industrial
Technology, 2Department of Biotechnology, Chung-Ang University
Recently, red seaweed attacks growing interests as 3rd
generation biomass due to their notable characteristics e.g., no
lignin, fast-growing rate, high contents of carbohydrates such
as cellulose and galactan. However, cellulose and galactan
should be degraded to mono sugar which will be used for the
fermentation to produce bioethanol. In this study, three fungal
strains with cellulose degrading activity were isolated. When
these fungus grown on cellulose culture the β-glucosidase
activity showed the highest. One of them was already
identified to phoma sp. and β-glucosidase from this fungus
purified to homogeneity. The purified β-glucosidase is a
tetramer protein of 440 kDa molecular weight and exhibits
optimal activity at 65°C, pH 4.5 The kinetic parameters, Km
and Vmax values with ρ-Nitrophenyl-β-D-glucopyranoside
(ρNPG) as a substrates are 0.21 mM and 113.87 μmol/min/mg,
respectively; with cellobiose the corresponding values are 0.14
mM and 78.55 mmol/min/mg. The activity is stimulated by
Mg2+, Zn2+ and inhibited by Fe2+, Cu2+.
[This work was supported by a grant from Korea Institute of
Energy Technology Evaluation and Planning, Ministry of
Knowledge Economy, Republic of Korea]
H003
H001
Chemoattractant-Mediated Rap1 Activation
Requires GPCR/G Proteins in Dictyostelium
Mannitol Accumulation in Fermentation of Kimchi
Using Leuconostoc Starters and Their Mutants
Taeck Jeon* and Injun Cha
Department of Biology, College of Natural Sciences, Chosun
University
Gan-Erdene Otgonbayar*, Seung Kee Cho,
Hwa Young Choi, and Nam Soo Han
Chungbuk National University, Department of Food Science and
Technology
Rap1 is rapidly activated upon chemoattractant stimulation and
plays an important role in cell adhesion and cytoskeleton
reorganization during chemotaxis. Here, we demonstrate that
G-protein coupled receptors and G-proteins are essential for
chemoattractant-mediated Rap1 activation in Dictyostelium.
The rapid Rap1 activation upon cAMP stimulation is absent in
cells lacking chemoattractant cAMP receptors cAR1/cAR3 or
a subunit of the heterotrimeric G-protein complex Gα2. Loss
of guanylyl cyclases GCA/SGC or a cGMP-binding protein
GbpC exhibits no effect on Rap1 activation kinetics. These
results suggest that Rap1, a key regulator for the regulation of
cytoskeletal reorganization during cell movement, is activated
through GPCRs cAR1/cAR3 and Gα2 proteins in a way
independent upon cGMP signaling pathway. F-actin polymerization is linked to Rap1 activation. In this study, we show that
inhibition of F-actin assembly result in extension of Rap1
activation kinetics, suggesting that F-actin assembly is required
for deactivation of Rap1 after chemoattractant stimulation.
[Supported by NRF grant funded by the Korean Government
(2009-0065992 and 2009-0070924)]
D-Mannitol, a six-carbon sugar alcohol, is about half as sweet
as sucrose and assumed to have several beneficial effects such
as an antioxidant and a non-metabolizable sweetener. Among
lactic acid bacteria only heterofermentative species are known
to convert fructose into mannitol. Mannitol production by
lactic acid bacteria in kimchi fermentation offers several
important advantages. This study is composed of two parts, at
first screening best mannitol producing wild type strains and
their EMS mutants secondly application of the promising
strains as starter culture for kimchi production. 8 heterofermentative lactic acid bacteria were compared as to their ability
to convert fructose to mannitol. Leuconostoc mesenteroides
9135, Leuconostoc mesenteroides 8293, and Leuconostoc
mesenteroides DRC strains were treated with EMS and were
grown with fructose addition. The yield of mannitol from
fructose was improved by 22%, 53%, 3% respectively.
Leuconostoc mutants were applied to kimchi as starter with
addition of 1%-3% fructose syrup and the mannitol
accumulation in kimchi was measured.
H002
H004
Understanding of Complex Nature of Evolution
Process Through Integrated Analysis of Multiple
Evolutionary Trees
*
Nutritional Requirements of Leuconostoc mesenteroides in a Chemically Defined Medium
Yujin Kim1*, DongYup Lee2, and NamSoo Han1
Department of Food Science & Technology, Chungbuk National
University, 2Department of Chemical & Biomolecular Engineering,
National University of Singapore, Singapore
Soon Ho Hong and Thuy Vu An Nguyen
School of Chemical Engineering & Bioengineering, University of
Ulsan
1
Evolution of living organism is combination of highly
complicated processes involving modification of various
features such as appearance, metabolism and sensing systems.
In spite of complex process of evolution, traditional evolutionary
analyses can only estimate single aspect of it. In this paper,
three evolutionary trees were constructed based on twocomponent system contents, metabolic network contents and
16S rRNA sequences. Then integrated analyses of trees were
carried out to understand various aspects of evolution process.
The results showed that integrated analysis can give new
insight into bacterial evolution study.
Leuconostoc species are heterofermentative gram-positive
bacteria that are commonly found in the fermented foods. They
play key roles in industrial and food fermentations. For
metabolic investigations, it is desirable to have a defined
growth medium for these bacteria which provides reproducibility of chemical composition; avoids an unnecessary excess
of the nutrients, facilitating the adjustment of their levels;
meets the experimentally determined nutritional requirements
of several strains. Therefore we developed a chemically
defined medium(CDM) for the growth of Leuconostoc
mesenteroides. The single-omission technique was applied to
each component of complete CDM in order to determine the
nutritional requirements. L. mesenteroides required glutamine,
isoleucine, methionine, valine, leucine, cysteine as essential
amino acids and threonine, phenylalanine, tyrosine, asparagine,
lysine, serine as additional amino acids for the growth. Glucose,
Mn, Mg, eight vitamins, adenine, uracil, and Tween 80 were
also consumed for the growth. This medium is simple and well
defined, and should be preferable to complex media for
conducting future biochemical, physiological, and genetic
studies.
[This work was supported by the Korean Systems Biology
Research Program (M10503020001-07N0302-00112) of
Ministry of Science and Technology (MOST).]
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H005
H007
Molecular Characterization of Lignin Degradation
Enzyme Genes for Pretreatment of Lignocellulosic
Biomass
*
Sun-Hwa Ryu , Myung Kil Cho, Bo-Young Kim,
Ji Yang, Mi-Hwa Choi, Myungkil Kim, and Sung-Suk Lee
Division of Forest Bioenergy, Korea Forest Research Institute
White rot fungi secret a large number of lignin enzymes such
as laccase, Mn-peroxidase(MnP) and lignin-peroxidase for
degradation of aromatic compounds in nature. Two laccase and
six MnP genes were isolated from Polyporus brumalis. The
expression of cDNAs was investigated with a view to
understanding the physiological functions of laccase and MnP
in relation to degradation of recalcitrant materials.The induction
of gene expression was stimulated by treatment of DBP.
Transcripts of pblac2, pbmnp2 and pbmnp4 were highly
expressed in the both of medium, whereas pblac1, pbmnp1 and
pbmnp5 genes were induced by DBP treatment. But the pattern
of total laccase and MnP enzyme activities were not exactly
coincided with that of the gene expressions. It can be
information that each laccase and MnP may have different
enzymatic properties and physiological functions in the
mechanism of DBP degradation. The identification of cDNAs
of laccase and MnP should contribute to the efficient
production of lignin degradation enzyme and the utilization of
basidiomycetous fungus for degrading lignin as well as many
recalcitrant xenobiotics.
H006
Identification of the Single-Nucleotide Polymorphisms
of COI in Crabs of the East Sea of Korea
Sun-Mee Hong*, Nyun-Ho Park, Dong-Goong Choi,
So-Jung Kim, Jung-Hee Woo, and Choong-Gon Kim
Department of Research and Development, Gyeongbuk Institute
for Marine Bioindustry
The aim of this study is to screen single nucleotide
polymorphisms (SNPs) of mitochondrial gene, COI, in crab in
the east sea of Korea. The cytochrome c oxidase (CO) plays
important roles in oxidative phosphorylation regulation and
oxygen sensing transfer. Defects involving genetic mutations
altering CO functionality or structure can result in severe, often
fatal metabolic disorder. In the present study, SNPs of
Cytochrome c oxidase subunit I (COI) were identified with the
techniques of sequence aligns and single-strand conformation
polymorphism (SSCP) in snow crab of Uljin, Pohang,
Youngduk and red crab in the east sea of Korea and Russia
crab.
In total, 12 SNPs were identified in the COI gene of the snow
crab including Russian crab, and red crab was defined for the 1
SNPs. This work will afford reference for the further study on
the association of COI with the adaptation to crab distribution
in the east sea of Korea.
[This study was supported by grant from MIFAFF.]
H008
Characterization of Diverse Lipolytic Enzymes
Derived from Marine Metagenomic Libraries
Accuracy Evaluation of Pyrosequencing for Microbial 16S rRNA Metagenome Analysis
Jeong Ho Jeon1,2*, Yun Jae Kim1, Hyun Sook Lee1,3,
Sang-Jin Kim1,3, Sang Ho Choi2, Sung Gyun Kang1,3, and
Jung-Hyun Lee1,3
1
Marine Biotechnology Research Center, Korea Ocean Research
& Development Institute, 2Molecular Microbiology and Toxicology,
Department of Agricultural Biotechnology, Seoul National
University, 3Department of Marine Biotechnology, University of
Science and Technology
Suk-Hwan Yoon1*, Soyeon Ahn2, Ok-Sun Kim1,
You-Seak Go3, Jeong-Sun Seo3,4, Taesung Park2,
Jonathan M. Adams1, and Jongsik Chun1
1
School of Biological Sciences and Institute of Microbiology,
Seoul National University, 2Department of Statistics, Seoul
National University, 3Macrogen, Inc., World Medridian Center,
4
Genomic Medicine Institute, Medical Research Center, Seoul
National University
Metagenomic libraries were constructed using marine sediments
from the deep sea of the Edison Seamount, the intertidal flat of
Ganghwa Island and the seashore of the Arctic station.
Seventeen positive clones were identified by screening for
lipolytic activity on tributyrin plates. Based on the phylogenetic
analysis, 13 enzymes were classified as belonging to Families I,
IV, and V, Feruloyl esterase group or Patatin-like protein
group, and 4 other enzymes were assigned to four new groups
that have never described. In an attempt to express the genes
with lipolytic activity as soluble proteins in Escherichia coli,
we employed a combination of approaches such as removing
the hydrophobic region in the N-terminus, co-expression of
chaperone genes and low temperature induction. Using affinity
chromatography, we obtained soluble proteins encoded by 14
genes. The optimum activities of the enzymes were determined
in the temperature range of 20–45°C and the pH range of 7.5–
9.5. Cold-activity was observed for 7 enzymes. While 13
enzymes showed esterase activity by hydrolyzing short-chain
fatty acid esters, EM3L4 showed lipase activity by hydrolyzing
long-chain fatty acid esters.
Pyrosequencing, one of the next generation sequencing
methods, generates massive sequence data with low cost
without clone library construction. At present, the most up-todate version, called 454 Genome Sequencer(GS) FLX
Titanium system, generates nucleotide sequence data with read
lengths of 400-500 bp on average. Use of pyrosequencing in
microbial 16S rRNA metagenome analysis has been developed
by several researchers and proved to be efficient in elucidation
of microbial community structure even of very complex
environments. However, the accuracy of the sequence data
from 454 GS FLX Titanium system has not yet been assessed
to date. We evaluated the accuracy of 454 GS FLX Titanium
by massive sequencing of 16S rRNA amplicons from fourteen
bacterial pure cultures. Sequencing by 454 GS FLX Titanium
produced reads with 51-547 bp in length, and the average
accuracy in raw data is 99.6%. In the analysis of comparisons
by read lengths, the higher accuracy rate was obtained in
longer read ranges. This study suggests that error rate of
pyrosequencing method is not substantially high enough for
hampering microbial 16S rRNA metagenome analysis based
on PCR of rRNA.
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H009
H011
Agreement, Precision and Accuracy of Epifluorescence Microscopy Methods for Enumeration of
Total Bacterial Numbers
1*
1
2
Eun Young Seo , Tae Seok Ahn , and Young Gun Zo
Department of Environmental Science, Kangwon National
University, 2Department of Biology, Kyungsung University
1
To assess interchangeability of estimates of bacterial abundance
by epifluorescence microscopy methods, total bacterial
numbers (TBNs) determined by widely accepted protocols
were statistically compared. Bacteria in a set of distinctive
samples were stained with acridine orange (AO), 4'-6diamidino-2-phenylindole (DAPI), and BacLight and enumerated
by visual counting(VC) and image analysis (IA). Model II
regression and Bland-Altman analysis proved general
agreements between IA and VC methods, although IA counts
tended to be lower than VC counts by 7% on a logarithmic
scale. Distributions of cells and latex beads on polycarbonate
filters were fitted to negative binomial models. The fitted
models revealed higher precisions of TBNs by the IA method
than those by the VC method. In pairwise comparisons of the
staining methods, TBNs by AO and BacLight staining showed
good agreement with each other, but DAPI staining had
tendencies of underestimation. The TBN values estimated by
AO and BacLight staining are relatively accurate and
interchangeable for quantitative interpretation and that IA
provides better precision than does VC.
H010
Rap1 Regulation of Actin Cytoskeleton in Dictyostelium
Taeck Jeon* and Injun Cha
Department of Biology, College of Natural Sciences, Chosun
University
Chemoattractant-mediated Rap1 activation helps establish cell
polarity by locally modulating cytoskeletons. Here, we
demonstratethat Rap1 interacts with RacGEF1 in vitro and
stimulates F-actin polymerization at the sites where Rap1 is
activated upon chemoattractant stimulation. Live cell imaging
using GFP-coronin, a reporter for F-actin, demonstrates that
cells expressing constitutively active Rap1 (Rap1CA) exhibit a
high level of F-actin uniformly distributed at the cortex
including the posterior and lateral sides of the chemotaxing cell.
Examination of the localization of a PH-domain containing
PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and
other Ras proteins in Rap1CA cells reveals that activated Rap1
has no effect on the production of PIP3 or the activation of
Akt/Pkb and Ras proteins in response to chemoattractant
stimulation. In vitro binding assay using truncated RacGEF1
proteins shows that Rap1 interacts with the DH domain of
RacGEF1, raising a possibility that Rap1 mediates F-actin
assembly by interacting with RacGEFI.
[This work was supported by National Research Foundation of
Korea Grant funded by the Korean Government (20090070924).]
H012
The Enhanced Half-Life of MxaJ Protein by Methanol Improves Methanol Dehydrogenase Activity
Regulation of Actin Cytoskeleton by Cortexillin
Through Arp2/3 Complex
Hee Gon Kim1*, Wonduck Kim2, and Si Wouk Kim1
Department of Environmental Engineering, BK21 Team for
Biohydrogen Production, 2Pioneer Research Center for
Controlling of Harmful Algal Bloom, Chosun University
Taeck Jeon* and Jisun Kang
Department of Biology, College of Natural Sciences, Chosun
University
1
A methanol dehydrogenase (MDH) is a key enzyme of
methanol oxidation process in Methylophaga aminisulfidivorans MPT. Previously, two types of MDH (MDH I and II) in
cell free extracts were purified from MPT cells grown on
methanol. The purified MDH I was confirmed to be composed
of two subunits (α, β MW = ~66, ~10 kDa) in a form of α2β2.
In addition, it was found that MDH II contained an additional
~30 kDa protein (MxaJ), designated γ, in a form of α2β2γ and
MDH II had 1.5 ~ 2.0 times higher activity than MDH I. In this
study, to investigate effect of methanol on MDH stability, the
half life of α and γ subunit of MDH in M. aminisulfidivorans
MPT grown on methanol and fructose was compared. To
obtain the half-life values, chloramphenicol was added into the
medium for translation halt. The analysis of each MDH subunit
analyzed by western blot indicates MxaJ protein from the
methanol grown cells was more stable than that from fructose
grown cells. The relative activity of MDH was proportional to
the amount of MxaJ protein. Based on these experimental data,
it appears that the γ subunit contributes to formation of active
MDH, thereby increases stability of MDH.
Cortexillin (Ctx) plays a critical role in the formation of cell
shape and reorganization of actin cytoskeleton in Dictyostelium. Here, we demonstrate that Ctx mediates reorganization
of actin cytoskeleton through Arp2/3 complex. Loss of Ctx
leads to a delayed chemoattractant-mediated ArpD translocation kinetics to the cortex with a peak at 8 sec, 2-3 sec slower
than in wild-type cells, indicating that proper translocation of
Arp2/3 complex to the cortex requires Ctx. Reduced cell
polarity and increased number of lateral pseudopodia in
chemotaxing cells lacking Ctx suggest Ctx’s functions in
controlling cell polarity and pseudopodia formation. In
chemotaxing cells, CtxI is enriched in the lateral sides of the
moving cells. Upon chemoattractant stimulation, there is a
rapid release of CtxI from the cortex followed by a transient
translocation to the cell cortex with a peak at -5 sec and a
subsequent decrease to basal levels. Highly dynamic subcellular
localization of CtxI implicates two more signaling components
mediating the translocation of Ctx during chemotaxis.
[This work was supported by National Research Foundation of
Korea Grant funded by the Korean Government (20090065992).]
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H013
Infectious Disease Biomarker Database (IDBD)
Kyung-Tae Jung, Kyenam Lee, Juhee Heo,
Hae-Seul Jeong, So-Jung Yoon, Cheon-Kwon Yoo, and
Kwang-Jun Lee
Division of High-Risk Pathogen Research, Center for Infectious
Disease, National Institute of Health, KCDC
For the biotechnological research revitalization and diagnosis
development of these infectious, We have constructed
Infectious Disease Biomarker Database (IDBD) by
knowledge-based web platform on useful biological contents
of pathogen biomarkers. IDBD provides several omics
information and global news of biomarkers as well as the
description of pathogens and disease. It is a community
annotation database utilizing collaborative Web 2.0 features,
providing a convenient user interface to input and revise data
online. It supports various types of data searches and
application tools to analyse sequence and structure features of
potential and validated biomarkers. Currently, IDBD integrates
710 biomarkers for 11 disease group, 79 infectious diseases
and 79 pathogens. Researchers can effectively utilize this
website as new information entry in the biotechnology field.
The content in IDBD is open and freely accessible to the
general public. (http://biomarker.cdc.go.kr) Also, we will link
biomarker information with pathogenic resource of national
culture collection for pathogen (NCCP) to distribution service.
H014
Korea National Microorganisms Research Resource
Center
Sang Seob Lee
Kyonggi University
The Korea National Microbiological Research Resource
Center is the core center of the twelve microorganism banks
designated by the Ministry of Education, Science and
Technology. The KNMRRC supports microorganism banks
with necessary guidelines, standards, training for efficient
operation of the banks. It also provides with an effective forum
to solve common issues of the related banks. The ultimate goal
of the KNMRRC is the followings: ①construction of
standardized
and
integrated
management
system,
②construction of Core center and other organs network,
③Quality Control(QC) of microbial resources in the member
banks, ④conservation of Resources in the member banks and
the interrupted banks, ⑤education for professionals in the
member banks, ⑥public Relations for raising people's
awareness of the importance of microbiological resources.
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Poster Sessions
H015
Korea National Environmental Microorganisms
Bank
Sang Seob Lee
Kyonggi University
Korea National Environmental Microorganisms Bank(KEMB)
has been established as a microbial and genetic resource center
for environmental industries. The KEMB plays an essential
role as follows: ①the collection and conservation of native
environmental microorganisms and genetic resources, ②the
construction of systematic management system for effective
conservation and application of microbiological resources for
environmental industries, ③the provision fundamental data
for ecosystem research and microbial classification, and ④the
development of biological treatment system for bioremedation
of environmental pollutant and ecosystem restoration. There
are about 14,000 strains of bacteria collected from
environments, at this time. These collections including
photosynthetic bacteria, oil-derived compounds removal
bacteria, industrial waste water disposal bacteria, odor
degrading bacteria and BOD/COD degrading bacteria are
classified in accordance with scientific and functional
characteristics, respectively. It is considered to promote
academic and industrial activities by supplying basic materials
for research and industrial applications, which accomplish the
ecological recovery through constructing eco-friendly
bioremediation system by supplying basic microbial resources.
H016
Center for Fungal Genetic Resources (CFGR):
Housing Plant Pathogenic Fungi For Educational
and Research Purposes
Yeo Kyoung Yoon* and Yong-Hwan Lee
Center for Fungal Genetic Resources, Seoul National University
Fungi are eukaryotic organisms, growing in a wide range of
habitats. Fungi are significantly important in a variety of ways.
They play an essential role in the decomposition of organic
matter. They have been used as a source of food, and agents for
fermentation of food products and for the production of various
antibiotics and enzymes that are used in a field of research,
industry, medicine, etc. In contrary, impact of many fungi on
animals and plants is economically and socially detrimental. For
example, Magnaporthe oryzae causes the most destructive
disease, “rice blast”. Annual yield loss of rice by rice blast is
equivalent to rice that could feed about 60 million people. The
Center for Fungal Genetic Resources (CFGR) was established
to collect, maintain and distribute genetic resources mainly from
plant pathogenic fungi, which are important for both educational
and research purposes. This will contribute to development of
new strategies for management of crop diseases and of new
components for improvement of our lives. CFGP possesses
important fungal species; a total of 35,400 isolates from 52
species of fungi including 21,090 T-DNA transformants of rice
blast fungus and anthracnose fungus. In addition to the
biological materials, CFGP has developed user-friendly
databases to maintain genetic information of fungal stocks and
help to solve questions about fungal pathogenicity, population
genetics, development, and evolution. Also, CFGP seeks
strategies for sustainable and scientific plant quarantine to better
protect our ecosystem from invasive microorganisms.
H017
H019
Bank of Waterborne virus
Culture Collection of Mushrooms
Soon-Young Paik
Department of Microbiology, College of Medicine, The Catholic
University of Korea
Cha Yoon Jeong, Jeong Hwa Kim, Jae Seong Lee,
Hae Jin Cho, Nuhu Alam, Ajith Indrawansha Rathnayake,
U Youn Lee, and Tae Soo Lee
Department of Biology, University of Incheon
The purpose of this bank is collecting and storing various
waterborne virus isolates provoking severe infections in animal
and human. This bank collects research information on various
viruses and provides this information when it is needed. We
provide various waterborne viruses, genomes and host cells to
hospitals, universities, research institutes, and government
institutes in the country as well as abroad. We also provide the
identification services of waterborne viruses and the research
data through on-line. Finally, it contributes to progress
biological science and to improve public health.
H018
The “Culture Collection of Mushrooms” (CCM) was founded
in 2008 and succeeded to Culture Collection of Wild
Mushroom Species which was established in 2002 at the
Department of Biology, University of Incheon, Korea. The
National Research Foundation of Korea (NRF) provides most
of the financial assistance for operation of CCM. All of the
mushroom cultures in CCM were preserved at 4°C refrigerator
or deep freezer of -80°C. The cultures in agar slant stored at
4°C were transferred periodically at the interval of 6 months.
The cultures stored in deep freezer were also transferred every
3 years. Since establishment of “Culture Collection of
Mushrooms”, CCM has been played supporting role for the
various research organizations by providing mushroom
cultures and mushroom DNA. CCM, also provides useful
information on mushroom researchers and industries. So far,
the CCM holds 3,700 mushroom strains which can be used for
basic and applied mushroom science research purposes. CCM
has been a member of World Federation for Culture Collection
(WFCC) since 2005. If anyone who are interested in
mushroom cultures and DNA from CCM, please contact
[email protected] or http://www.wildmush.or.kr
H020
Phagebank
Korea Bank for Pathogenic Viruses
*
KyoungEun Cha and Heejoon Myung
Department of Bioscience and Biotechnology, Hankuk University
of Foreign Studies
Bacteriophages are viruses growing on bacterial hosts. They
are antagonistic to bacteria and first reported by Frederick
Twort and Felix d'Herelle in 1915 and 1917, respectively.
They are found in sea, air, land and even foods. It is assumed
that 1030 to 1032 phages exist on earth and they play a role in
maintenance of biological balance. Recently, new applications
for phages are increasingly reported. As they are a part of
useful biological resources, there are increasing demand for
secure these resources. In response to these demands, this bank
collects phages from environments as well as from working
groups worldwide. Currently, 50 different phages are stocked.
At the same time, we serve as a distributor for the collected
phages.
Ki-Joon Song
Korea Bank for Pathogenic viruses
Korea Bank for Pathogenic Viruses in Korea(KBPV;
www.kbpv.co.kr) has been established in 2005 as a repository
agent for the collection, management and distribution of the
various pathogenic viruses that are essential to use for
researches in biomedical sciences. The Institution operates in
collaboration with The Institute for Viral Disease at
Department of Microbiology, College of Medicine, Korea
University, founded in 1973. The bank has unique viral
collections such as Hantaan, Seoul, Muju, and Soochong, the
etiologic agents of hemorrhagic fever with renal syndrome. To
date, total of more than 43,000 materials(~100,000vials) from
human and animal have been collected and maintained. We
have provided a highly collaborative environment for
researchers in various fields by providing valuable viral
resources including consulting service. We also provide the
educational program related to pathogenic viruses including
biosafety training.
Requestors of such agents are required to register with
KBPV and to supply details of their laboratory facilities and
safety management. More details about KBPV can be found at
http://www.kbpv.co.kr.
205
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H021
H023
Plant Virus GenBank
Myxobacteria Bank
Ryu Ki Hyun
Department of Horticultural Science, Seoul Women's University
Jinwoo Chung and Kyungyun Cho*
Myxobacteria Bank, Department of Biotechnology, Hoseo
University
Plant Virus GenBank (PVGB) is a nonprofit semi-governmental
organization, one of the Korea National Research Resources
Collections (KNRRC) for special research materials Banks program
financially supported by the Ministry of Education, Science &
Technology (MEST) dedicated to collection, identification, characterization, preservation, research development, distribution and deposition
of plant virus research biomaterials established since 1999. PVGB is
one of substructure of Korea National Microbiological Research
Resources Collections (KNMRRC). PVGB retains a number of
accessions and a wide range of collections of Plant Virus Biomaterials
useful for Plant Virology and Biotech-related research areas. PVGB
has moved to its current status on November in 2000 and has modern
facilities and infrastructures for supporting broad research fields as well
as Plant Virology Community. PVGB has been recognized as a
member of World Federation for Culture Collection & World Data
Center for Microorganisms (WFCC-WDCM) and ISBER since April
of 2001 and June of 2007, respectively. Main objectives and contents
of PVGB can be categorized as 7 topics as follow ; collection and
development of Plant Virus Research Biomaterials such as infectious
plant virus culture, plant viral cDNA clone, plant virus antiserum,
biologically active full-length cDNA clone, viral cDNA library, virusinduced plant cDNA library, and diagnostic primers, preservation of
Plant Virus Research Biomaterials, Distribution of Plant Virus
Research Biomaterials to worldwide researchers to support their
research fields and Safe Deposit from virologists, Development of New
Plant Virology Techniques, i.e., molecular taxonomy of plant viruses,
infectious cDNA clones, molecular indexing of virus variation,
screening of virus resistance, virus-resistant transgenic plants, and risk
assessment for living modified (LM) virus and LM plant systems,
collection and support of Research Information of Plant Virology, and
workshop and seminar for Plant Virology.
H022
Culture Collection of Antimicrobial Resistant
Microbes
Yeonhee Lee*, Eunju Shin, Hakmi Lee, Kieun Lee,
Hyunjin Hong, Hyeran Nam, Miyoung Lee, and Jihye Park
Culture Collection of Antimicrobial Resistant Microbes,
Department of Biology, Seoul Women’s University
Antimicrobial resistance became a world-wide problem and
this must be dealt via cooperation among microbiologist,
biochemist, medical doctor, pharmacologist, agricultural
scientists. Since Culture Collection of Antimicrobial Resistant
Microbes was established in 1999, CCARM has been played a
role as a connector among various research fields by providing
the antimicrobial resistant microbes with known mechanism
and information. CCARM collects, keeps, and preserves the
resistant microbes in a systemic manner for constant supply of
certified microbes and share the information with researchers
in various fields. To date, CCARM has a collection of over
19,000 strains of bacteria and yeast from 87 genera and
provides various information including international meeting,
newest information related to resistance via homepage and
newsletter. CCARM is now increasing the interaction and
collaboration between culture collections through national and
international network as a member of Clinical Laboratory
Standards Institute since 2000, World Federation for Culture
Collection & World Data Center for Microorganisms since
2003, International Society of Biological and Environmental
Repositories since 2007, Korea National Research Resource
Center since 2008, and Biological Repositories since 2009.
206
Poster Sessions
Myxobacteria—gram-negative soil bacteria belonging to δproteobacteria—produce a wide variety of bioactive secondary
metabolites and enzymes with unique action mechanisms.
However, in comparison with other microorganisms such as
actinomycetes, relatively few myxobacterial strains have been
isolated; moreover, the biological activities of their metabolites
and enzymes have not been studied extensively. Thus,
myxobacteria are considered as one of the last microbial
resources for identifying new medicines, enzymes, and
biopolymers. In the light of this situation, Myxobacteria Bank
has been appointed as one of the National Research Resources
Banks by the Korean Science and Engineering Foundation in
2007. Currently, Myxobacteria Bank maintains a collection of
2,451 wild myxobacterial strains isolated in Korea and 488
culture extracts, 40 genomic DNA, and 48 clones of secondary
metabolite biosynthetic genes obtained from these strains. The
bank also has 34 myxobacteria type strains and genetic tools
used to manipulate myxobacteria.
H024
Lichen as a Novel Bioresources in Korea
Jae-Seoun Hur and Young Jin Koh
Korean Lichen Research Institute, Sunchon National University
Lichens are symbiotic organisms composed of a fungus
(mycobiont) and an alga (photobiont). They produce
characteristic secondary metabolites, lichen substances, which
seldom occur in other organisms. Lichen and their metabolites
have many biological activities. In spite of the wide spectrum
of biological activities shown by the lichens, they have long
been neglected by mycologists and overlooked by agrochemical industry because of its slow growth in nature and
difficulties in the artificial cultivation of organisms. Use of
lichen-forming fungi can overcome the disadvantage of natural
lichen extracts for industrialization of their metabolites because
of their much faster growth and larger production of the
metabolites in culture than the natural thalli. Korean Lichen
and Allied Bioresources Center focuses on isolation, maintenance and distribution of lichen bioresources to research
groups in universities, national institutes and industrial sectors.
It also screens their biological activities, and investigates
cultural conditions for large production of lichen substances.
Chemical library of some lichen extracts is also available from
the center.
H025
Korea Marine Microalgae Culture Center
Sung Bum Hur
Department of Marine Bio-materials and Aquaculture, Pukyong
National University
Today microalgae are widely used in research and as educational materials.
They are also commercialized in the industries of food, animal feed and
environment. Microalgae exhibit a promising potential to be converted into
pharmaceutical products and bio-fuel energy. For this reason, there are
active, ongoing researches on microalgae with tremendous expectations of
scientists The Korea Marine Microalgae Culture Center (KMMCC) was
established with a financial support from Korea Science & Engineering
Foundation. In 1995, the collection of microalgae strain has been increasing
continuously since 1995, and its number reached to about 1,600 strains in
2010. The collection mainly consists of marine strains (83%) which are
mostly isolated from Korean waters (89%). The major classes of the strains
are Bacillariophyceae (57%), Chlorophyceae (20%), Dinophyceae (5%),
Cyanophyceae (5%), Prasinophyceae (4%), etc. With respect to
identification of the strains, about 52% and 44% of them are identified at the
level of species and genus, respectively. In addition, 4% are unidentified,
and about 56% of the strains are under axenic state. The culture strains of
the KMMCC are introduced regarding information on sampling, culture,
biological and chemical characteristics of each strain. The initial direction of
the KMMCC focused on finding microalgal strains that had a good dietary
value for larvae in aquaculture. Such strains used to be supplied to the
hatchery. Our recent work, however, has shifted to collecting a wider range
of diverse microalgae which are taxonomically different. In accordance with
the KMMCC's progression on the strain collection, the demand for the
strains in research fields has been expanding from the industry of
aquaculture to biotechnology, environmental sciences, engineering,
biological oceanography, etc. in Korea. Recently, the annual domestic
request for the algal strains from academic and commercial organizations
has increased up to nearly 100 requests for as many as 200 strains. Making a
deposit with newly isolated microalgal strains in the KMMCC by other
investigators is always possible if they agree to be open about distributing
their strains to other researchers as well. The final decision of the strains to
be deposited belongs to the KMMCC. We have diversity isolated more than
1,600 monospecific strains mainly from Korean coastal waters. Even
though assigning a correct taxonomical position to the strains has always
been our primary concern, many difficulties still exist in identifying them.
H026
Helicobacter pylori Korean Type Culture Collection
(HpKTCC) Collects and Distributes Clinical Isolates
of H. pylori
Hyung-Lyun Kang1,2, Myung-Je Cho1, Jae-Young Song1,2,
Woo-Kon Lee1, Seung-Chul Baik1,2, Kon-Ho Lee1,
Kwang-Ho Rhee1,2
1
Department of Microbiology, Gyeongsang National University
School of Medicine, 2Helicobacter pylori Korean Type Culture
Collection, Gyeongsang National University School of Medicine
H. pylori that colonizes only in human gastric mucosa is one of
the most common human pathogens and is the main cause of
gastritis, peptic ulcer, and gastric cancer. Despite the clinical
and commercial importance of H. pylori, many researchers
have been blocked to investigate the diagnosis, treatment, and
prevention of H. pylori infections because of difficulty in
obtaining H. pylori isolates from patients. We have collected
and characterized H. pylori isolates obtained from worldwide
areas to allow researchers to access a variety of characterized
H. pylori isolates. characterized H. pylori isolates. H. pylori
KTCC contributes to promote the study for the diagnosis,
treatment, and prevention of H. pylori infections by providing
fundamental research materials to investigators.
207
www.msk.or.kr
2010 International Meeting of
the Microbiological Society of Korea
Author Index
209
www.msk.or.kr
210
Author Index
A
Abell, Guy
Adams, Jonathan M.
Ahn, Hye-Ran
Ahn, Jae-Hyung
Ahn, Jae Hyoung
Ahn, Joong-Hoon
Ahn, Keug-Hyun
Ahn, Soyeon
Ahn, Tae-Seok
Alam, Nuhu
Algire, Mikkel
An, Dong-Shan
An, Ji Eun
An, Kwang-Deuk
An, Sang Eun
Anandham, Rangasamy
Atomi, Haruyuki
B002
H008
B039
B010
G001
B029
E014, E016
H008
B004, B006, H009
H019
S9-3
C026
A028
B032
F016
A042
S10-1, S10-3
B
Bae, Dongryeoul
C1-3
Bae, Hee Sun
C021
Bae, Hye-Jin
S9-5, F037
Bae, Jin-Woo
S1-2, A001, A002, A005, A006, A007,
A008, A010, A026, A027, A032, B002,
B005, B033, B045, B048
Bae, Jung Eun
G001, G002
Bae, Kyung Sook
A009, A011, E001, E002
Bae, Songmee
D025, D026
Baek, Hyun
B039
Baek, In-Joon
S8-2, F018
Bahk, Jang-Jun
B040
Bahn, Young-Ho
S6-1
Baik, Seung-Chul
H026
Bak, Eun Jung
E005, E006
Bak, Sun Uk
E012
Bang, Duhee
S9-2
Bat-Ochir, Chinbayar
F031
Bok, Jung-Im
A036
C
Cha, Chang-Jun
Cha, Injun
Cha, Jaeho
Cha, Jeong-Heon
Cha, Ju-Hee
Cha, KyoungEun
Cha, Sang-Ho
Cha, Sun-Shin
Chae, Ae-Rhee
Chae, Chang-Suk
C025, G015
H001, H011
F013
D009, D011, E005, E006
C025
H018
D014
S10-4
F027
S6-5
Chae, Ji-Hwa
Chae, Jong-Chan
Chae, Suhn-Kee
Chai, Young Gyu
Chang, Ho-Won
Chang, In-Ho
Chang, Kyung-Soo
Chang, Yeon-Ji
Chang, Young-Hyo
Chen, Wilfred
Chen, Xiaoqiang
Cheong, Chaejoon
Cheong, Soon-Gee
Cho, Eun-Jung
Cho, Ahnna
Cho, Eun-Min
Cho, Eun Ji
Cho, Ga Youn
Cho, Hae Jin
Cho, Hee-Jung
Cho, Hyun-Hee
Cho, Jae-Chang
Cho, Jae-Yong
Cho, Jang-Cheon
Cho, Kyeung Hee
Cho, Kyung-Ju
Cho, Kyungyun
Cho, Myung-Je
Cho, Myung Kil
Cho, Nam Hyuk
Cho, Sang-Buem
Cho, Seong Kee
Cho, Seung-Hyon
Cho, Seung Kee
Cho, Su-Hee
Cho, Ye-Seul
Cho, Yong-Hyun
Cho, Yoo-Bok
Cho, You-Hee
Cho, Youn-Dong
Cho, Yun-Seok
Choa, Yong-Ho
Choe, Gyeong-Im
Choe, Senyon
Choi, Ah Reum
Choi, Ahyoung
Choi, Cheong-Up
Choi, Chi Won
Choi, Da Mi
Choi, Dong-Goong
Choi, Eun Ju
Choi, Ho Gil
Choi, Hong-Jae
Choi, Hwa Young
G002
C004
F030, F031
A019, D020
B033
B004, B006
D006, D007
E031
C018
C010
S11-1
S12-2
S9-5
S2-4
B004
F014
F023
S13-1
H019
D029
B025, B046
B022, B023, B024
F008, G003
A013, A035, B050, E020, G007
B026
C001
H023
H026
H005
S3-4
D032, D033, D034, G014
F007
S2-1, E033, E034
C001, F010, H003
B038
F027
C1-2, D005
F009, F012
S11-1
D027
B038, B039
C010
D008, D012, D013, D014
S12-2
E020
A013
D008, D012, D013, D014
B044, D035
G001
H007
C014
E005
B027
C003, F007, H003
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Choi, Hyo-Suk
B055
Choi, Hyoung T
E009
Choi, Hyoung Tae
E010, E011, E012, E013, E035
Choi, Hyun-Ah
D005
Choi, Jae-Yu
D027, D029
Choi, Ji-Hyeon
G004
Choi, Jin Hee
B021
Choi, Jiyoung
A012, C008, C013
Choi, Jong-Soon
S10-2, E015
Choi, Jongwoon
C008, C013, E017
Choi, Jung-Hoon
E018
Choi, Jung-Hye
A027
Choi, Jung Youn
G015
Choi, Kieun
A018
Choi, Kyoung-Hwa
F013
Choi, Mi-Hwa
H005
Choi, Myung Sik
S3-4
Choi, Nack-Shick
E014, E016
Choi, Sang Ho
H006
Choi, Sang Ki
C020
Choi, Seung-Ik
B006
Choi, Sunga
C008, C013, E017
Choi, Ung Kyu
C021
Choi, Youngcheol
A012
Choi, Yusang
D015, D016, D017, D018, D019
Chokesajjawatee, Nipa
S1-4
Choo, Jinho
F021
Choo, Yeon-Sik
S13-5
Chowdhury, Wasimul B.
C024
Chu, Hyuk
S3-4
Chun, Jeong-Hoon
D028
Chun, Jong Woo
C006
Chun, Jongsik
W1, A017, B009, B035, H008
Chung, In-Sik
D009, D011
Chung, Jin Woong
S11-4
Chung, Jinwoo
H023
Chung, Sanghoon
C008, C013
Chung, Won-Yoon
E006
Chung, Young-Ho
S10-2, C1-1, B044, E015
Cui, Yidan
D001
D
Dong, Ke
Donohue, Tim
Dufour, Yann
Dwidar, Mohammed
C017
F012
F012
C009
E
Efkaen, Sisi Patricia
Ekpeghere, K. I.
Eom, Chi-Yong
Eom, Hyun-Ju
212
Author Index
G012
B034
F014
F010
Eom, Jeong Seon
F024, F025
F
Franks, Ashley E.
Fujiwara, Shinsuke
B007
S10-3
G
Geum, Neri
Ghim, Sa-Youl
Go, You-Seak
Goh, Ah-Ra
Gwak, Joo-Han
C006
A015, A016, C011, C012
H008
C020
S9-5, F038
H
Ha, Changwan
Ha, Nam-Chul
Ha, Sang-Jun
Ha, Un-Hwan
Hahm, Mi-Seon
Hahn, Yoonsoo
Hahn, Youn-Soo
Ham, Su-Jin
Han, Ah-Reum
Han, Gui Hwan
Han, Nam Soo
D015, D016, D017, D108, D019
F005, F006
S6-1
F003
C012
B045
D021
E001, E002, E003
E022
B018
A028, C001, C003, C019, E004,
F007, F010, H003, H004
Han, Song Ih
B042
Han, Sun-Hee
B020
Hanh, Nguyen Phan Kieu
F026
Heo, Dong-Hyuk
S8-2
Heo, Eun-Jung
B027
Heo, Juhee
D036, H013
Ho, Cuong T.
B019
Hong, Hyunjin
H022
Hong, Jeoung-Hyun
C002
Hong, Jin-Kyung
B022
Hong, Jin Pyo
B056
Hong, Kee-Jong
S6-3
Hong, Seok-Hyun
F009
Hong, Seung-Beom
S5-3
Hong, Soon Gyu
A035, B026, C005, G013
Hong, Soon Ho
H002
Hong, Sun-Mee
G008, H007
Huh, Won-Ki
E019, E021, E031, E032
Hur, Hor-Gil B003, B008, B019, B029, C010, C015, G006
Hur, Jae-Seoun
A023, A024, A034, H024
Hur, Sung Bum
H025
Hur, Yoon-Ki
D029
Hwang, Eunha
S12-2
Hwang, Gui-Hye
G003
Hwang, Hee Jung
D008, D012, D013, D014
Hwang, Hyun-Gook
Hwang, Jae Sung
Hwang, Jae Yoon
Hwang, Ji-Sun
Hwang, Jooyea
Hwang, Jung-Sook
Hwang, Soo-Myung
Hwang, Sungmin
Hwang, Ui Wook
Hwang, Ye-Ji
Hwang, Yong-Il
Hwang, Young-Ok
Hyun, Ryu Ki
C020
G010
F026
S6-5
D008, D012, D013, D014
S13-5
D007
F013
S4-3
A016, C012
E014
G007
H021
I
Im, Choon-Soo
Im, Sin-Hyeog
Im, Su-jin
Imanaka, Tadayuki
Inoue, Kengo
Itoh, Takashi
A030
S6-5
D015, D016, D017, D018, D019
PL1, S10-1, S10-3
B007
S1-3
J
Jang, Hyoung Ryoul
Jang, Il-Gyo
Jang, Jeonghwan
Jang, Jong-Ok
Jang, Jung Im
Jang, Sungil
Jang, Woo-Yeong
Jang, Woo-Yeoung
Jang, Yu Ji
Jeon, Byoung Seung
Jeon, Byung-Seung
Jeon, Che Ok
Jeon, Dong In
Jeon, Dusik
Jeon, Hyun Bum
Jeon, Jeong Ho
Jeon, Seon-Ae
Jeon, Seon Hyun
Jeon, Taeck
Jeon, Young Ho
Jeong, Cha Yoon
Jeong, Eun Kyo
Jeong, Gil-Sang
Jeong, Gilhwa
Jeong, Hae-Seul
Jeong, Hyun-Jeong
Jeong, Je Yong
Jeong, Jung Sun
Jeong, Seong-Yoep
B057
A029
B003
B021
F024, F025
D009, D011, E006
S9-5
F038
A045
A014, A018
C009
B045, B048
A019
C008, C013
A012
H006
A015, A016
G002
H001, H011, H012
S12-2
H019
G001, G002
A012
C008, C013, E017
D036, H013
A035
B053
G002
E014, E016
Jeong, Yoonhwa
Jeong, Yun-Hee
Jhun, Min A
Ji, Chang-Jun
Jiang, Cheng-Lin
Jiang, Shenghua
Jin, Seon-Yeong
Jin, Xiao Ling
Jo, Eun-Hye
Jo, Gyeong-Jin
Jo, Hyun-Jung
Jo, Jae-Hyung
Jo, Sung-Kee
Joa, Jae-Ho
Joh, Kiseong
Jones, Kathleen R.
Joo, Young Min
Joshi, Yogesh
Joung, Yochan
Jung, Byung Hoon
Jung, Da-I
Jung, Dawoon
Jung, Gyoo Yeol
Jung, Jae Sung
Jung, Jae Young
Jung, Jaejoon
Jung, Ji-Hee
Jung, Ji Young
Jung, Jia
Jung, Kwang-Eun
Jung, Kwang-Hwan
D021
S6-1
E032
S2-2
S1-1
C010
B027
F006
A043
C002
D006
F022
G005
B049
A003, A004
D009, D011
D009, D011
A023, A024, A034
A003, A004
E027
E016
B004
S9-1
A038, A039, A040, B036
E027
B001
C014
B045, B048
B026
E007
S8-3, E020, E023, E024, E025,
E026, E027, E028
Jung, Kyoung Hwa
A019, D020
Jung, Kyung-Ju
D019
Jung, Kyung-Tae
D036, H013
Jung, Mi-Ja
A001, A002, A005, A006, A007, A008,
A026, A027, A032
Jung, Su-Jin
F011
Jung, Won Seok
F035
Jung, Yong-Gyun
F009, F012
Jung, You-Jung
B004, B006
Jung, Young-Mi
D021
K
Ka, Jong-Ok
Kahng, Hyung-Yeel
Kamashita, Tomoyuki
Kanai, Tamotsu
Kandori, Hideki
Kang, Beom Sik
Kang, Dae-Ook
Kang, Dae Woo
Kang, Hyun Ah
A043, B047, B049
A033, A038, A039, B036, B037
S10-3
S10-1, S10-3
S12-5
S12-1
E016
F025
F020, F032, F033
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Kang, Hyun Bae
Kang, Hyung-Lyun
Kang, Ji-Hyun
Kang, Jisun
Kang, Junghwa
Kang, Kook Hee
Kang, Seung-Gu
Kang, Sung Gyun
Kang, Tae-Sook
Kang, Yeonho
Kang, Yu Ri
Kato, Chiaki
Kefala, Georgia
Khan, Sumera Afzahl
Kigawa, Rika
Kim, B.-H.
Kim, Baek Joong
Kim, Bo-Young
Kim, Byoung-Chan
Kim, Byung-Chun
Kim, Byung-Yong
Kim, Cha-Yeon
Kim, Chang-Jin
Kim, Chang Jeon
Kim, Choong-Gon
Kim, Chul-Joong
Kim, Chun Sung
Kim, Dae Hyun
Kim, Do Young
Kim, Dockyu
Kim, Dong-Hun
Kim, Donghwan
Kim, Donghyun
Kim, Eun Hye
Kim, Eung-Soo
Kim, Eungbin
Kim, EunJae
Kim, Hae-Seon
Kim, Haemin
Kim, Hak Jun
Kim, Han-Suk
Kim, Han Bok
Kim, Hanuel
Kim, Hee-Jeong
Kim, Hee-Kyoung
Kim, Hee Gon
Kim, Hong-Man
Kim, Hong-Mi
Kim, Hongik
Kim, Hwang Yeon
Kim, Hyangmi
Kim, Hye-Jin
Kim, Hye Won
Kim, Hye Young
214
Author Index
S3-5
H026
G007
H012
D003
D021
B016
E015, G007, H006
A045
D025, D026
E010
B046
S12-2
S13-5
B032
B034
E011
H005
S1-5
A011, A031
A043
F027
S1-1, A020, A021, A022, B021
B053, B054, B055, B057
C002, F004, G008, H007
C018
D022, D023
C021
E001, E002, E003
B017, C005
B008
C007
E017
A035, B026, F032
G013
B017
G004
E007
C004
S12-3
S3-2
G010
A003, A004
D006, D007
F023
C016, H010
F005, F006
D028
C007, D021
G010
A009, A011
B024
E011, E012
E013, E035
Kim, Hyun-Jin
S2-1, E030, E033, E034
Kim, Hyun-Jung
D010, D028
Kim, Hyunjung
B015
Kim, Ik Sang
S3-4
Kim, In Seop
G001, G002
Kim, Inhae
S9-1
Kim, Insook
F015
Kim, Jae-Jin
A044
Kim, Jae-Myung
D008, D012, D013, D014
Kim, Jaisoo
B015, B016, B051, C017, D005, D010
Kim, Jaisoo Su
C1-2
Kim, Jandi
A032
Kim, Jee-Young
B015
Kim, Jeong-Hee
D030
Kim, Jeong-Yoon
F020, F032, F033
Kim, Jeong Hwa
H019
Kim, Ji Eun
C003, E004
Kim, Ji Sun
A028
Kim, Jihong
C008, C013, E017, F017
Kim, Jihyun F.
E024, E026
Kim, Jin Seok
F024, F025
Kim, Jin Young
G001
Kim, Jinmoon
E005, E006
Kim, Jong-Guk
S13-5
Kim, Jong-Shik
S4-4, C002, F004, G008
Kim, Jong Soo
S12-3
Kim, Jonggill
A012
Kim, Joy (Hyun)
S2-5
Kim, Ju-Young
F036
Kim, Jun-Tae
F004, G008
Kim, Jung-Hoon
S2-2
Kim, Jung-Wan
E018, E022
Kim, Jungok
G006
Kim, Kang-Lim
D007
Kim, Ki-Yeon
G011
Kim, Kwang-Wook
S3-5
Kim, Kwang Kyu
A041
Kim, Kyoung-Ho
S1-2, B002, B005, B033
Kim, Kyung-Duk
B010
Kim, Kyung-Mi
B011
Kim, Kyung Jin
S8-1
Kim, Kyungsun
B017
Kim, Laurentius Jongsoon
D006
Kim, Mi-Ree
B043
Kim, Mi-Seon
S4-2
Kim, Mi Na
A031
Kim, Min-Gyu
C010
Kim, Min-Joo
D006
Kim, Min-Ju
S8-4
Kim, Min-Sik
F009, F012
Kim, Min-Soo A001, A002, A005, A006, A007, A010,
A026, A027, A032, B005, E014, E016
Kim, Min Jee
F032, F034
Kim, Min Jung
D024
Kim, Mincheol
Kim, Minyoung
Kim, Miri
Kim, Moon Chang
Kim, Myungkil
Kim, Na-Yeon
Kim, Nam-Shik
Kim, Oh Cheol
Kim, Ok-Sun
Kim, Ok Bin
Kim, Sae-Hun
Kim, Sang-Dal
Kim, Sang-Jin
Kim, Sang-Yoon
Kim, Sang Hoon
Kim, Sang Jin
Kim, Sang Wook
Kim, Sanguk
Kim, Se-Il
Kim, Se Jun
Kim, Sehwan
Kim, Seil
Kim, Seon-Won
Kim, Seung Bum
Kim, Seung Il
Kim, Si Wouk
Kim, Sin Yeon
Kim, So-Jung
Kim, So-Yeon
Kim, So-Young
Kim, Song-Gun
Kim, Soo-Jin
A017, B009
E005, E006
S2-1, E033, E034
E014
H005
C002
D021
F033
B009, B035, H008
E008
D002, D003
S13-5
A025, B043, B046, G007, H006
F008
A019, D020
C1-1
S3-4
S9-1
C009
E025
E026
A014, A018
F036
B056
S10-2, C1-1, B044, D035, E015
B018, C016, H010
F029
H007
B026
C001, E023
C026
S5-3, A036, A037, A042,
A043, B047, B049
Kim, Soo-Ki
D032, D033, D034, G014
Kim, Soo-Kyoung
D015, D016, D017, D018
Kim, Su-Ok
G014
Kim, Sun-Ho
F016
Kim, Sung-Kee
C017, D010
Kim, Sung-Tae
C011
Kim, Wan-Gyu
B047
Kim, Wha-Jung
C011
Kim, Wonduck
C016, H010
Kim, Yi-Na
C020
Kim, Yi-Seul S5-3, A036, A037, A042, A043, B047, B049
Kim, Yong-Jae
F003
Kim, Yong Jin
F016, G015
Kim, Yoo-Taek
B016
Kim, You Sun
E024
Kim, Young-Chang
S9-5, F037, F038
Kim, Young-Joo
C014
Kim, Young-Mi
B012
Kim, Young-Pil
S12-2
Kim, Young Bong
D027, D029
Kim, Young Min
F019
Kim, Younghoon
D002
Kim, Yu-Kon
B055
Kim, Yu Jin
C001, C003, H004
Kim, Yun-Ji
A006
Kim, Yun-Jung
D033, D034
Kim, Yun Jae
H006
Kim, Yun Jeong
C006, D030
Kim, Yunmin
B009
Kishore Babu, Bandamaravuri
B018
Kiyuna, Tomohiko
B032
Klammt, Christian
S12-2
Koh, S.-C.
B034
Koh, Young Jin
A023, A024, A034, A040, H024
Koo, Chang-Duck
B030
Kook, Joong-Ki
D022, D023, D024
Krock, Hans J.
S13-4
Ku, Hyun-Ok
D013
Kum, Hyun-Woo
E009
Kwak, Jun-Yong
F030
Kwon, Hak Cheol
C014
Kwon, Ho-Keun
S6-5
Kwon, Hyeok-Do
B013
Kwon, Kae Kyoung C1-1, A025, B040, B043, B044, B046
Kwon, Min Sik
C023
Kwon, Ohsuk
F020, F032, F033, F034, F035, F036
Kwon, Sang Oh
B044, D035
Kwon, Soon-Kyeong
E024, E026
Kwon, Soon-Wo
S5-3, A036, A037, A042,
A043, B047, B049
Kwon, Suk-Tae
S10-5
Kwon, Tae-Hyung
G008
L
Lee, Byung Soo
Lee, Chan-Gul
Lee, Chang-Muk
Lee, Chang-Ro
Lee, Changhan
Lee, Cho Hee
Lee, Choong-Gu
Lee, Don-Hwa
Lee, Dong-Heon
Lee, Dong-Hun
Lee, Dong Gyu
Lee, Dong Hyuck
Lee, Dongsun
Lee, DongYup
Lee, Eun-Gyeong
Lee, Eun-Joo
Lee, Eun-Sook
Lee, Gun-Seung
Lee, Hae Ran
Lee, Hak-Sung
S3-5
G014
B049
S2-1, E030, E033, E034
F015, F017
G010
S6-5
S4-2
A033, A038, A039, B036, B037
B030, B031
B044
G001
S12-4
H004
F035, F036
B027
B020
B020
B042
D011
215
www.msk.or.kr
Lee, Hakmi
H022
Lee, Han-Ki
D021
Lee, HanBoreum
C021
Lee, Hee-Jung
D029
Lee, Hong Kum
A035, B026, C005
Lee, Hun-Goo
E031
Lee, Hyang Burm
B053, B054, B055, B056, B057
Lee, Hye-Jung
B016
Lee, Hyi-Seung
S13-3
Lee, Hyo-Jin
B041, D007
Lee, Hyo Jung
B045, B048
Lee, Hyoung Ro
A028
Lee, Hyun-Ju
C017, E001, E002, E003
Lee, Hyun-Jung
C1-4
Lee, Hyun Sang
C006
Lee, Hyun Sook
H006
Lee, Hyune Hwan
F022
Lee, In-Jung
S13-5
Lee, In-Koo
S13-5
Lee, Insun
C018, D021
Lee, Jae-Chan
A020, A021, A022, B021
Lee, Jae Ho
F019
Lee, Jae Il
G001, G002
Lee, Jae Seong
H019
Lee, Jaehoon
D025, D026
Lee, Jang-Hyun
D027
Lee, Jeong Im
G001, G002
Lee, Ji-Youl
C1-2, D005
Lee, Jin-Mok
S8-4
Lee, Jin-Uk
C002
Lee, Jin-Won
S2-2
Lee, Jin-Woo
B040
Lee, Jin Ha
B051
Lee, Jin Sung
A044, G013
Lee, Jinho
G012
Lee, Jong Kyu
S12-3
Lee, Joon-Hee S11-3, D015, D016, D017, D018, D019
Lee, Jung-Hyun
A025, B040, E015, H006
Lee, Jung-Sook
S5-2, A041
Lee, Junghoon
E029
Lee, Kang-Mu
C024, D001, D002, D031
Lee, Kang Hyun
A009, A011, A031
Lee, Kangmu
D003
Lee, Kangseok
F005, F006
Lee, Keon Ah
E028
Lee, Keun Chul
A041
Lee, Ki Young
A033, E020
Lee, Kieun
H022
Lee, Kihyun
B009, B035
Lee, Kon-Ho
H026
Lee, Kui-Jae
C004
Lee, Kun-Jae
C010
Lee, Kwang-Jun
D036, H013
Lee, Kyenam
D036, H013
216
Author Index
Lee, Kyu-Ho
S3-2
Lee, Kyung-Chang
F011
Lee, Lan Hee
E001, E003
Lee, Minho
F005
Lee, Miyoung
H022
Lee, Mok-Young
B020
Lee, Myunju
B031
Lee, Oh Hyoung
A045
Lee, Sang-Hoon
B023
Lee, Sang-Seob
S5-1, C1-2, B015, B016, B051, C017,
D005, D010, H014, H015
Lee, Sang-Yual
B014
Lee, Sang Yup
F035, F036
Lee, Seung-Cheol
S7-4
Lee, Seung-Ju
C1-2
Lee, Sun Bok
A033
Lee, Sung-Jae
A012
Lee, Sung-Suk
E009, H005
Lee, Sung Gu
E035
Lee, Sung Hyuk
A025
Lee, Sungkyoung
D025, D026
Lee, Tae Soo
H019
Lee, U Youn
H019
Lee, Won-Ho
F038
Lee, Woo-Kon
H026
Lee, Woong
S4-2
Lee, Yeol Gyun
E015
Lee, Yeon Ju
S13-3, E022
Lee, Yeonhee
H022
Lee, Yin-Won
F023
Lee, Yong-Hwan
H016
Lee, Yong Heon
F029
Lee, Yong Hoon
C004
Lee, Yong Joo
S8-1
Lee, Yoo Kyoung
A035
Lee, Young-Eun
C002
Lee, Young-Hee
D034
Lee, Young-Hyuk
B055
Lee, Young-Ok
B027
Lee, Young Sun
A038, A039, A040, B036
Lee, Yun-Jung
D032, D033, D034
Lee, Yung Mi
B026, C005
Li, Wen-Jun
S1-1
Lim, Gwang-Hui
B027
Lim, Gyu-Bum
E019
Lim, Hyang Soon
E011
Lim, Jae-Hong
C010
Lim, Jeesun
C024, D001, D002, D003, D004
Lim, Jeongheui
C018, D021
Lim, Young Woon
A044
Liu, Fang
C010
Lory, Stephen
PL2
Lou, Kai
S1-1
Lovley, Derek R.
B007
Luo, Dan
Luo, Yuan Rong
G009
C1-1
M
Maeng, Pil Jae
Margesin, Rosa
Maslennikov, Innokentiy V.
Matsui, Toshiro
Merrell, D. Scott
Min, Kyung-Bae
Min, Mun-Kyoung
Misawa, Norihiko
Moon, Chuloo
Moon, DongGeRaMi
Moon, Eun Young
Moon, Hye-Yun
Moon, Jin Seok
Mun, Hye Yeon
Mun, Mi Ran
Murtas, Giovanni
Myung, Heejoon
Myung, Nosang V.
S8-1, F016
B026
S12-2
S7-2
S3-3, D009, D011
S9-5, F037
F034
S13-2
B010
F028
A035
F020
C001
B053, B054, B055, B057
C018
S9-4
H018
C010, G006
N
Nakanosho, Masahiro
Nam, Doo Hyun
Nam, Gi-Baeg
Nam, Hyeran
Nam, Ji-Hyun
Nam, Jong Hee
Nam, Seong-Won
Nam, Young-Do
Nedashkovskaya, Olga I.
Nguyen, Anh Dung
Nguyen, Huan H.
Nguyen, Thi Thuy
Nguyen, Thuy Vu An
Noh, Hyun-Ju
Noh, Hyung-Jun
S10-3
F026
F012
H022
B030, B031
S6-5
S11-1, D001, D004
A001, A002, A005, A008,
B002, B005, B033
A025
A024
S6-2
A024
H002
G013
B049
O
Oh, Byung-Taek
Oh, Doo-Byoung
Oh, Hee-Bok
Oh, Hyun-Myung
Oh, Hyun-Woo
Oh, Jae Sung
Oh, Jeong-Il
Oh, Kye-Heon
Oh, Kyong-Hee
C004
F032, F033, F034, F035, F036
D028
A013, B050, E020
A009, A011
A039
S8-4, E007
B037, B038, B039
B031
Oh, Kyoung Hee
Oh, Min-Kyu
Oh, Yu-Kyung
Ohkuma, Moriya
Olsen, Cara H.
Otgonbayar, Gan-Erdene
A044
A018
D029
B032
D009, D011
H003
P
Paik, Hyun-Dong
Paik, Soon-Young
Pak, Jae-Hong
Park, Ah Reum
Park, Chae Haeng
Park, Chan-Hee
Park, Chan-Sun
Park, Chan-Won
Park, Chankyu
Park, Dong-Jin
Park, Doo-Sang
Park, Eun-Jin
Park, Ha-Young
Park, Ha Ju
Park, Hae Youn
Park, Hee-Moon
Park, Ho-Yong
Park, Hyo-Jin
Park, In-Cheol
Park, Jae-Won
Park, Jae-Yeon
Park, Jae-Yoon
Park, Jang Min
Park, Jeong Mi
Park, Ji Hyun
Park, Jihye
Park, Jin-Soo
Park, Jin-Sook
Park, Jong-Tae
Park, Jong Myong
Park, Joo-Hong
Park, Joon-Seok
Park, Jungsoon
Park, Kwang-Jin
Park, Kyung Sun
Park, Nokyoung
Park, Nyun-Ho
Park, Sae Woong
Park, Seong-Kyu
Park, So Young
Park, Su-Jin
Park, Sun Jue
Park, Sun Wook
Park, Sung-Jin
D027
H017
S4-2
G015
G013
S6-1
E014, E016
B049
E029, F015, F017
A020, A021, A022, B021
A009, A011
S1-2, A001, A005, A006, A008,
A026, A032, B048
D015, D016, D017, D018, D019
C005
B017
A009
E001, E002, E003
S6-1
S5-3, A037
D032, G014
C009
D022, D023, D024
B018
F007
D020
H022
C014
A029, A030, B025
E022
A015
F012
S6-1
B028
S8-4
S12-3
G009
C002, F004, G008, H007
F019
A001
A012
D015
E013
E035
C011
217
www.msk.or.kr
Park, Sungsu
S11-1, C024, D001, D002, D003, D004
Park, Taesung
E032, H008
Park, Woojun
S11-2, B001, B028, F001, F002
Park, Yong-Ha
C018, D021
Park, Yong Keun
F024, F025, F029
Park, Yong Sin
A045
Park, Yongjin
C024, D031
Park, YongKeun
G004
Park, Young-Hyun
B036
Park, Yu Ri
C016
Park, Yun-Seon
F028
Peck, Kyong-Ran
C1-2, D005
Peterkofsky, Alan
S2-1, E034
Piao, Shunfu
F005, F006
Pinhassi, Jarone
G007
R
Rathnayake, Ajith Indrawansha
H019
Rejish Kumar, V. J
C018, D021
Rhee, Joon Haeng
S6-5
Rhee, Kwang-Ho
H026
Rhee, Kwang-Hyun
B050
Rhie, Gi-Eun
D028, D030
Rho, Hyun Soo
S4-1
Roe, Jung-Hye
F009, F011, F012
Roh, Changhyun
G005
Roh, Seong Woon
A001, A002, A005, A006, A007,
A008, A010, A026, A027, A032, B002, B033
Roh, Young Hoon
G009
Rung, Doo Il
E035
Ryu, Choong-Min
C012
Ryu, Jae-Ha
S7-3
Ryu, Ji-Young
B029
Ryu, Sun-Hwa
E009, H005
S
Sadowsky, Michael J.
Sahoo, Anupama
Sang, Byoung-In
Sano, Chie
Sato, Takaaki
Sato, Takako
Schinner, Franz
Seo, Byeong Joo
Seo, Eun-Young
Seo, Hyun-Seok
Seo, Jeong-Sun
Seo, Sang Woo
Seok, Soon-Ja
Seok, Yeong-Jae
Shahid, Sudipto
Shik, Chun-Shik
218
Author Index
B029, C010
S6-5
A014, A018, B010, C009, G011
B032
S10-1
B046
B026
C018, D021
B006, C003, C019, H009
A025
H008
S9-1
S5-3
S2-1, E030, E033, E034
A019, D020
E031
Shin, Eunju
H022
Shin, Hee-Sung
F003
Shin, Hee Jae
S13-3
Shin, Ji-Hye
B031
Shin, Kee-Sun
A005, A008, A011, A027, A031
Shin, Mun-Sik
S9-5, F037, F038
Shin, Na-Ri
A002, D030
Shin, Sung Jae
S3-5
Shin, Sunme
B017
Shon, Min jeong
F020
Shon, San Ho
A038, A039
Sim, Sangjun
C008, C013
Sim, Se-Hoon
F005, F006
Sin, Yesl
C007
So, Byung-Jae
D008, D012, D013, D014
So, Jae-Seon
S6-5
Sohn, Hosung
S3-5
Sommer, Peter
S6-4
Son, Ji Hyun
C006
Son, Jung-A
A043, B047, B049
Son, Kwang-Hee
E001, E002, E003
Son, Su-Wan
C1-2
Song, Hong-Gyu
B011, B012, B013, B014
Song, Hye Jung
G010
Song, Jae-Young
H026
Song, Jae Jun
F034
song, Jung-A
E017
Song, Ki-Joon
H020
Song, Kiwon
S2-3
Song, Kyung
C010
Song, Saemee
F006
Song, Yong Bhum
E032
Song, Yu-Jin
B038
Song, Yun-Jin
B039
Sugiyama, Junta
B032
Suh, Yaesul
B003
Sumayo, Marilyn
C012
Sung, Chang-Keun
B021
Sung, Hye-Ri
A015, A016
Surh Young-Joon
S7-1
Suwannarangsee, Surisa
F033
T
Takedomi, Shogo
Takemasa, Ryo
Tang, Shu-Kun
Thawng, Cung Nawl
Tomita, Junko
Tran, Hoa
S10-3
S10-1
S1-1
C015
B032
B007
U
Ueki, Toshiyuki
B007
Um, Youngsoon
Unden, Gottfried
Unno, Tatsuya
A014, A018, B010, C009, G011
E008
B003
V
Van, Nguyen Dinh
D027
W
Wang, Chinling
Wang, Tianshi
Wang, Yun
Weon, Hang-Yeon
Whang, Kyung-Sook
Whitmire, Jeannette M.
Whon, Tae Woong
Won, Choul-Jae
Won, Mi-Young
Woo, Eun Ju
Woo, Jung-Hee
Woo, Sung-Il
C1-3
E018
S1-1
S5-3, A036, A037, A042,
A043, B047, B049
B041, B042
D011
A008
S3-5
G014
A044
F004, G007, G008, H007
D021
X
Xu, Li-Hua
Xu, Yongbin
S1-1
F005, F006
Y
Yang, Eun-Bin
Yang, Hee-Jong
Yang, Hyun Ok
Yang, Jae-Seong
Yang, Ji
Yang, Ki-Hoon
Yang, Seung-Jo
Yang, Seung-Joon
Yang, Sujeong
Yang, Sung-Hyun
Yeo, Kwonjoo
Yeom, Gyoseon
Yeom, Jinki
Yi, Dae-Gwan
Yi, Hana
Yim, Joung Han
Yokooji, Yuusuke
E021
E014, E016
C014
S9-1
H005
E007
B050
C001
B015
A025
S12-2
C022, C023
S11-2, F001, F002
E021
B007
C005
S10-1
Yoo, Cheon-Kwon
Yoo, In-Hwa
Yoo, Ji-Sun
Yoo, Ki-Seon
Yoo, Seung-Hee
Yoo, Yun-Jung
Yoon, Byoung-Dae
Yoon, Byung-Dae
Yoon, Gun-Mook
Yoon, Jang W.
Yoon, Jeong-Jun
Yoon, Jin Ho
Yoon, Jung-Hoon
Yoon, Juyoung
Yoon, Mi Young
Yoon, Min
Yoon, Sang Sun
Yoon, Seo-Yeon
Yoon, So-Jung
Yoon, Suk-Hwan
Yoon, Tae-Young
Yoon, Won Suck
Yoon, Yeo Kyoung
You, Won-Sun
You, Yung-Hyun
Youn, Min
Yu, Dong-Joo
Yu, Jaeyon
Yu, Ji-Eun
Yu, Kijong
Yu, Seungho
Yu, Yeong Man
Yun, Bong-Sik
Yun, Cheol-Won
Yun, Dae-Myoung
Yun, Hye-Ok
Yun, Ji-Hyun
Yun, Seulgi
Yun, Sung-Hwan
Yun, Sung Ho
D028, D030, D036, H013
F003
F012
C003
A042
D009, D011, E006
E016
E014
C001
D002
G015
F027, F028
A015, A016
S11-1
B052, C024, D031
D001, D004
S3-1, B052, C024, D031
B030
D036, H013
B009, H008
C022, C023
G004
H016
D036
S13-5
D002
G002
D025, D026
S9-5, F037
C007
B031
F016
C004
S8-2, F018
C022, C023
B055
A005
F035
F023
C1-1, B044, D035
Z
Zhang, Li-Li
Zhao, Li-Xin
Zhi, Xiao-Yang
Zhou, Peng
Zo, Young-Gun
S1-1
S1-1
S1-1
B007
S1-4, H009
219
www.msk.or.kr
124
Colloquium